Microporous materials, methods of making, using, and articles thereof

ABSTRACT

Described herein are methods for separating one or more analytes present in a fluid sample. The methods involve passing the fluid through or into a microporous material, wherein the analytes are localized near the surface of the microporous material. Additional processing steps such as hybridization and amplification can be performed once the analyte is localized. In one method, once the analyte is localized, the analyte can be detected, counted, and correlated in order to determine the concentration of the analyte in the sample. In another method, the localized analyte is destabilized to make the localized analyte more accessible for chemical manipulation. Modified microporous materials and composite materials are also disclosed that can be used in any of the methods and articles described herein. The composite is composed of a microporous material and a pigment, wherein the pigment is incorporated in the microporous material. The pigments alter the optical properties of the microporous material, which enhances the detection of analyte once it is localized. Methods for making pigmented composites are also disclosed. In a further aspect, various kits and articles such as filtration devices containing any of the microporous materials described herein are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of International ApplicationNo. PCT/US2003/037598, filed on Nov. 20, 2003, which claims the benefitof U.S. Provisional Application Nos. 60/429,093 and 60/429,259, bothfiled Nov. 26, 2002. These applications are hereby incorporated by thisreference in their entireties for all of their teachings.

BACKGROUND

The quantification and identification of analytes present in samplesderived from a subject is important in the medical arena. In particular,the quantification and identification of analytes from a sample cangreatly assist in the diagnoses and treatment of numerous diseases.

For example, nucleic acid analysis is a widely used technique in medicaldiagnostics. Current techniques of nucleic acid analysis routinelyquantify several hundred molecular copies of target nucleic acids permilliliter of patient sample. This requires substantial molecularmultiplication, often through a polymerase chain reaction (PCR), toachieve sufficiently high nucleic acid concentrations for detection withconventional laboratory equipment. This molecular amplification requiresnucleic acid purification prior to multiplication, which is timeconsuming, expensive, difficult to control, and has limited accuracy.The final assay result is highly dependent on closely controllingseveral multistep processes. Currently, available viral load assaysreport log (rather than linear) values of nucleic acid concentration, atleast in part due to limited precision and accuracy inherent in currenttechniques.

The amplification techniques described above require purified nucleicacid samples free of competing or interfering contaminants. Contaminantscan be present in the sample and may include enzymes, proteins,hemoglobin, bacteria, particulate, etc. Alternatively, contaminants cancome from the purification system used and may include organic solvents,salts, metal ions, etc. Removing contaminants that interfere withnucleic acid amplification (reverse transcription, amplification, etc.)as well as removing contaminating substances that interfere withdetection (hybridization, fluorescence, etc.) requires undertakingtime-consuming processes.

Techniques exist to purify nucleic acids. For example, multiple organicreagent extraction/precipitation using chloroform, phenol, and loweralcohols have been used for many years to isolate and purify nucleicacids for subsequent analysis. Additional purification techniquesinclude chromatographic techniques using ion-exchange chromatography,reversed phase chromatography, affinity chromatography, and variouscombinations thereof.

Silica based nucleic acid purification techniques are currently in widecommercial use. These systems purify nucleic acids through reversiblebinding (precipitation) onto silica and its derivatives in the presenceof chaotropic agents and/or organic solvents. Typically, these systemsdilute patient samples containing nucleic acids into 5+ molar guanidinethiocyanate (GTC). This mixture reacts at room temperature, then equalvolumes of neat ethyl alcohol are added and vortexed. This mixture isexposed to high surface area silica, where the nucleic acids bind almostimmediately. The silica is recovered, rinsed several times withsolutions containing GTC and ethyl alcohol, dried, and then the boundnucleic acids are eluted with low salt water. Several versions of thistechnique exist, and are differentiated based on the silica-based binderused. For instance, one system uses a small filtration or spin columncontaining a thick silica fiber mat for binding. Other systems use glassbeads, magnetic silica based beads, silica impregnated filters, etc.These systems add a carrier nucleic acid (co-precipitant) to permitisolation and purification of very low concentrations of target nucleicacid. While silica based nucleic acid purification techniques adequatelyperform their intended function, these techniques are time-consuming andexpensive.

As with purifying nucleic acids, many techniques exist for immobilizingnucleic acids. To facilitate nucleic acid capture, surfaces are oftentreated with a variety of chemicals that bind to nucleic acids when thesurface is contacted with a solution containing the nucleic acid. Forexample, covalent attachment of organic compounds to glass and aluminumoxide is known. Such attachment may involve the use of silanizationreagents to completely modify the native oxide surface for control ofcharge, hydrophobic effects, etc., as well as to permit covalentattachment of nucleic acids, proteins, polymers, etc. to effect captureof specific target molecules to the surface. Quartz, glass, and siliconsubstrates with various surface chemistries have been used to capturenucleic acids for optical molecular detection. These techniques requiretarget nucleic acids to diffuse to the reactive surface forimmobilization or capture. At low concentrations, thesediffusion-controlled reactions require hours to complete. Thesetechniques are time-consuming, expensive, and may not be suitable forrapid optical molecular detection techniques.

Accordingly, it would be desirable to have materials, devices, andmethods for the rapid, inexpensive, and efficient isolation of ananalyte from a sample. It is also desirable to remove any contaminantsthat may interfere with further manipulation (e.g., quantification andidentification) of the analyte once it has been removed from the sample.

SUMMARY

Described herein are compositions, methods, devices, and machines forseparating one or more analytes present in a fluid sample. In oneaspect, the method involves passing the fluid through a filter composedof a microporous material, wherein the analytes are localized near thesurface of the microporous material. Additional processing steps such ashybridization and amplification can be performed once the analyte islocalized on the microporous material. In one aspect, once the analyteis localized on the microporous material, the analyte can be detected,counted, and/or correlated in order to determine the concentration ofthe analyte in the sample having a known volume.

In another aspect, composite materials are disclosed. In certainembodiments, these composites can be used in any of the methods andarticles described herein. The composite is composed of a microporousmaterial and a pigment, wherein the pigment is incorporated in themicroporous material. The pigments alter the optical properties of themicroporous material, which enhances the detection of analyte once it islocalized near the surface of the composite.

In another aspect, modified microporous materials are disclosed composedof a microporous material and a suspension matrix, wherein thesuspension matrix is localized near the surface of the microporousmaterial.

In a further aspect, various kits and articles such as filtrationdevices containing any of the microporous materials or as describedherein are provided.

The advantages of the microporous materials, methods, articles, andmachines described herein will be set forth in part in the descriptionwhich follows, or may be learned by practice of the aspects describedbelow. The advantages described below will be realized and attained bymeans of the elements and combinations particularly pointed out in theappended claims. It is to be understood that both the foregoing generaldescription and the following detailed description are exemplary andexplanatory only and are not restrictive.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several aspects described below.It will be appreciated that these drawings depict only typicalembodiments of the microporous materials, articles, and methodsdescribed herein and are therefore not to be considered limiting oftheir scope.

FIG. 1 a is a top-view of the microporous material. FIG. 1 b shows thecross-sectional view of the microporous material.

FIGS. 2 a and 2 b show the localization of an analyte near the surfaceof a microporous material.

FIG. 3 shows a fluorescent bead based tracer process for a localizedanalyte.

FIG. 4 shows the use of enzyme linked fluorescence for the detection ofa localized analyte.

FIG. 5 shows a graph of the timing of a typical ELF process using thetechniques described herein.

FIG. 6 is a graph showing the fluorescent spot count versus time fornucleic acid counting.

FIGS. 7 a-7 c depict modified microporous materials and their use forenhanced reaction rates.

FIG. 8 depicts a device for the detection of localized analytes.

FIG. 9 depicts an alternate device for the detection of localizedanalytes.

FIG. 10 depicts an alternate device for the detection of localizedanalytes.

FIG. 11 shows human genomic DNA localized on a nickel/boron Anoporesurface and stained with Syber Gold dye.

FIGS. 12 a-12 h depict processed, negative images of the YOYO-1 labeledcalf thymus DNA that was optically detected.

FIG. 13 is a graph of an uncorrected nucleic acid counting (NAC) assaycalibration curve.

FIG. 14 is a graph showing the corrected nucleic acid count with respectto nucleic acid concentration.

FIG. 15 depicts a filtration device for the localization of an analyte.

FIG. 16 depict an alternate filtration device for the localization of ananalyte.

FIG. 17 depict an alternate filtration device for the localization of ananalyte.

FIG. 18 depict an alternate filtration device for the localization of ananalyte.

FIG. 19 depict an alternate filtration device for the localization of ananalyte.

FIG. 20 depict an alternate filtration device for the localization of ananalyte.

FIG. 21 depict an alternate filtration device for the localization of ananalyte.

FIG. 22 depict an alternate filtration device for the localization of ananalyte.

FIG. 23 depict an alternate filtration device for the localization of ananalyte.

FIGS. 24 a and 24 b are graphs of nucleic acid retention vs. NaClconcentration for plain unmodified, diol (acid hydrolyzedglycidoxypropyltrimethoxysilane) modified, andamine(aminopropyltrimethoxysilane) modified 0.2 micron Anoporemembranes.

FIGS. 25 a and 25 b are graphs of nucleic acid retention vs. saltconcentration (chaotropic and NaCl) for plain unmodified Anoporemembranes.

FIG. 26 is a graph of nucleic acid retention vs. pH for plainunmodified, diol modified, and amine (APS) modified 0.2 micron Anoporemembranes.

FIG. 27 is a graphs showing the nature of nucleic acid retention on aplain unmodified Anopore membrane in variable NaCl, with 1 mM Trisbuffer, pH 8.0.

FIG. 28 is a graph showing the nature of nucleic acid retention on anamine (APS) modified Anopore membrane in variable NaCl, with 1 mM Trisbuffer, pH 8.0.

FIG. 29 is a graph showing the nature of nucleic acid retention on adiol modified Anopore membrane in variable NaCl, with 1 mM Tris buffer,pH 8.0.

FIG. 30 is a graph showing the effect of phosphate buffer concentrationon nucleic acid retention or elution for plain unmodified and amine(ethylenediaminopropyltrimethoxysilane, EDAPS) modified membranes.

FIG. 31 is a bar graph of a sample digestion/filtration study forcerebral spinal fluid (CSF), human serum, and whole blood.

FIG. 32 is a photograph of the electrophoretic gel showing ampliconsgenerated by PCR multiplication of beta globin target regions present onthe localized genomic DNA contained within the whole blood samples.

FIG. 33 is a real time amplification curve for P globin PCRamplification/detection of nucleic acids from lysed blood.

FIG. 34 shows P globin amplicon melting curves of nucleic acids fromlysed blood.

FIG. 35 is a graph of scan time vs. spot dimension for five detectionconditions.

FIG. 36 is a graph for the conditions of 100 dye molecules/nucleic acidat various spot dimensions, and between 2 and 500 signal-to-noise ratio(SNR).

FIG. 37 is a graph for the conditions of 100 dye molecules/nucleic acidat various spot dimensions, and between 2 and 100 SNR.

FIG. 38 is a graph for the conditions of 25 dye molecules/nucleic acidat various spot dimensions, and between 2 and 500 SNR.

FIG. 39 is a graph for the conditions of 100 dye molecules/nucleic acidat various spot dimensions, and between 2 and 100 SNR.

FIG. 40 is a high magnification negative image photomicrograph of lambdaphage DNA of 48,502 base pair length localized on an unmodified 0.2micron pore size Anopore membrane filter and stained with YOYO-1 dye.

FIG. 41 shows optical absorption versus wavelength for three organic dyemodified Anopore membranes and one Anopore membrane withnickel-phosphorous) deposited on the membrane (the deposited pigmentmembrane).

FIG. 42 is an electron micrograph of the interior of an Anopore membranemodified with nickel-boron.

DETAILED DESCRIPTION A. Compositions and Methods for Separating andAnalyzing Analytes

There are many situations in which it is desirable to be able toseparate or analyze an analyte from or in a given sample. An analyte canbe any target substance, which is to be analyzed, for example, in asample. For example, in disease diagnoses, often there is a particularanalyte that is produced by, or part of, a pathogen, and physicians usethe presence or absence of that analyte to determine whether the patientis infected with the pathogen. There are many characteristics andparameters that affect this type of determination, such as theefficiency of detection, how sensitive the detection methods are, theamount of analyte produced by the pathogen, the amount of sample neededto perform the analysis, the stability of the analyte, the amount ofbackground analyte present in the sample, and so forth. Methods andcompositions which increase the sensitivity of detection and the easewith which an analyte can be detected are desirable.

Typically the methods involve manipulating the analytes in sample, byfor example, separating the analytes from the sample, localizing theanalytes, analyzing the analytes, by for example, counting the number ofanalytes separated, correlating the number of analytes counted to thenumber of analytes in the sample, purifying the analytes, and collectingthe analytes, for example, after one or more other manipulationactivities are completed. These steps and others can be performed oneafter the other or many or all of them can be performed together.

Described herein are methods for separating one or more analytes presentin a sample, such as a fluid sample. Typically the fluid sample willhave a known volume, which facilitates certain types of analysis of theanalyte. “Separating an analyte from a fluid sample” means removing theanalyte from the sample, such as the fluid sample. The amount andprecision of analyte separation from the sample can vary. In one aspect,qualitative determination for the presence or absence of the analyte maybe performed on less precise and less efficient analyte separation fromthe sample. In another aspect, analyte is separated with increasedprecision and efficiency. In this aspect, the separated analyte can besubjected to further manipulation, such as quantification discussedherein. The purity of the separated analyte will also vary, ranging fromvery pure to an analyte containing impurities from the sample.Purification of at least 2, 5, 10, 25, 50, 75, 100, 150, 250, 500, 750,1,000, 5,000, 10,000, and 25,000 fold relative to the starting samplecan be achieved.

In one aspect, the method involves passing the sample, such as the fluidsample, through a filter. Typically the filter will have or be made froma microporous material. When the sample is passed through the filter,the analytes can be localized near the surface of the filter. Thedisclosed filters and microporous materials can have many differentsurfaces, such as a planar surface, an external surface, an internalsurface, a wetted surface, and a channel surface. Often the analytes arepassed through a microporous material. A microporous material is anymaterial having a plurality of pores, holes, and/or channels. Themicroporous material permits the flow of liquid through or into thematerial.

In certain embodiments the materials are localized and this localizationis reversible, and in other embodiments the localization isirreversible. The localization can also include the characteristic ofbeing immobilized, which means that the analyte is retained in a definedarea long enough to be analyzed, for example, counted or assayed.

The characteristics of the filter and the microporous material affectthe parameters of separating and/or manipulating the analyte in thesample. In certain embodiments the microporous material is a compositeor modified microporous material, and these composites are discussedherein. Often it is desirable to detect the analytes that have beenlocalized, by for example, viewing them directly or assaying for sometype of label that has been associated with the analyte. This has beentraditionally performed by what may be termed an analog signal detectiontechnique, where all analytes contribute to an “ensemble” signal that isrepresentative of the number of analytes localized to the microporoussurface. Additionally, the techniques disclosed herein allow for whatmay be termed digital detection or analyte molecular counting. Asdiscussed in further detail below, molecular counting of the analyte isaffected by a number of different parameters including, the actualdetectable signal from the analyte molecule or label and the amount ofbackground signal observed by the detection method. Furthermore, thereis a threshold level of signal that is required for detection and thisthreshold level is different for each detection method used. Thus, to beable to “count” a particular analyte that has been localized on theplanar surface of a filter, the absolute detectable signal from theanalyte molecule or label that is localized needs to be high enough sothat there is signal observed from the analyte that is above thebackground signal for the detection method, and which is above theabsolute threshold for signal detection or the analyte could belocalized but not counted. As discussed herein, any of the microporousmaterials described herein can be used in filters to decrease thebackground signal produced by the filter for a particular detectionmethod. For analog detection, this decrease in background signal allowsfor the detection of fewer absolute numbers of an analyte because asmaller signal is required from the analytes to be observed over thebackground signal. For digital detection such as molecular counting,this decrease in background signal allows better resolution ofindividual molecules and greatly improves molecular detection. Thus, thedisclosed methods and compositions allow for greater sensitivity inanalyte detection, i.e. they allow for detection of smaller numbers ofanalyte in a sample. This is desirable because it allows for use ofsmaller samples and tests to identify the presence or absence of ananalyte and may be more sensitive and accurate, for example. Asdiscussed herein, in certain embodiments the composites utilize apigment to decrease the background signal.

Once the analyte has been localized near the surface of the microporousmaterial, such as a composite or a modified microporous material,further processing steps may be performed. The analyte can also be, forexample, amplified, detected, or isolated. For example, the analyte canalso be counted, correlated, purified, or collected.

The microporous materials, methods, articles, and kits described hereinprovide improvements over conventional methods for the separation andanalysis of analytes, and are more simplified than current techniques.Additionally, the microporous materials, methods, articles, and kitsdescribed herein are useful either individually as separate steps ormaterials or methods individually or as combinations of the disclosedmaterials and method steps. For example, the ability to rapidly isolatenucleic acids from a wide variety of impure substances quickly andsimply through a microporous material is useful regardless of whetherthese isolated nucleic acids are subsequently counted.

Further discussion of the various parts of the disclosed articles andtypes of compositions and microporous materials, as well as varioussteps and sets of methods steps are set forth in more exemplary detailbelow.

B. Localization of Analyte

The disclosed methods generally are used for localizing an analyte in asample. There are many different types of samples that can be used thatcan be a source of the analyte.

1. Samples

The samples are typically fluids because a fluid facilitates passagethrough the disclosed filters or microporous materials, for example.Sample fluids containing analytes include solutions or suspensions, suchas solutions of molecularly dissolved materials or hydrodynamicallysuspended materials. Sample fluids that contain analytes includebiofluids, such as whole blood, serum, plasma, cerebral spinal fluid,urine, saliva, semen, sputum, bronchalveolar lavage fluid, jointaspirate, or wound drainage. Other sample fluids that can be usedinclude various preparations containing bacteria, viruses, fungi,spores, cell cultures, fecal excrements, animal tissues or cells,vegetable tissues or cells, lysed ingredients thereof, or combinationsthereof. It is understood that a solid sample containing an analyte canbe homogenized or otherwise put into solution to facilitate the analysisof the sample.

In another aspect, the source of the analyte can be an environmentalsample. For example, waste water containing contaminants such aspolymers or other chemicals. In another aspect, the sample could be abiowarfare sample, which is considered a sample that has beenpotentially contaminated with a biowarfare agent. For example, abiowarfare sample could be a water sample, such as a potable watersample, that may have been contaminated. In another aspect, the samplecan be an air sample.

In another aspect, the sample can be a fluid for human consumption suchas, for example, drinking water and other beverages.

i. Known Volume Samples

Often the samples will be utilized in a known volume condition. A knownvolume sample is one in which the volume or amount of sample is known,Typically it is important to use known volume samples if one wishes todetermine the concentration of analyte present in the sample at somepoint during the process, but also importantly if the amount of analytethat is present in the sample is to be correlated to the amount ofanalyte in the organism from which the sample was obtained. For example,if one desires to know how many viral particles are present in asubject, a sample, such as a blood sample can be taken from the subject.This blood sample can be analyzed using the disclosed methods andcompositions and the amount of analyte present in the sample can bedetermined. To determine how much analyte was present in the subject,one needs to determine how much analyte was present in the sample, in aknown volume, and then extrapolate this to the amount of analyte presentin the subject based on the knowledge, for example, of the total volumeof, for example, the fluid in the subject.

The size of the known volume can depend on for example, the amount ofanalyte in the sample, the sensitivity of detection of the analyte, orthe types of manipulation planned for the analyte. For example, when thesample is a blood sample, the known volume can be less than 40 μl, lessthan 30 μl, less than 20, less than 10 μl, or less than 5 μl for genetictesting, while samples of plasma or CSF for viral load testing may begreater than 200 μl, such as 200 μl to 10,000 μl. In another aspect, theamount of sample that contains the analyte is from 0.5 μl to 1,000 μl,0.5 μl to 900 μl, 0.5 μl to 800 μl, 0.5 μl to 700 μl, 0.5 μl to 600 μl,0.5 μl to 500 μl, 0.5 μl to 400 μl, 0.5 μl to 300 μl, 0.5 μl to 200 μl,0.5 μl to 100 μl, 0.5 μl to 50 μl, 0.5 μl to 25 μl, or 0.5 μl to 10 μl.In another aspect, when the sample is an environmental sample such aswater, air, etc., the volume of the sample can be larger than 1milliliter, such as 10 mL to 1,000 mL.

2. Analyte

As indicated the disclosed compositions and methods are typicallydesigned to manipulate analytes, for which information is desired. Anyanalyte that has the properties necessary for localization on themicroporous material or that can be bound to a suspension matrix forlocalization, can be targeted or manipulated.

Numerous analytes can be localized near the surface of any of themicroporous materials described herein. For example, the analyte can bea protein, a parasite, a fungus, an effector molecule, a ligand, areceptor, a signal-generating molecule, a structural molecule, an ion,an antigen, an antibody, a tissue, a cell, a bacterium, a protein, or acombination thereof. Additionally, analytes include any target moleculesthat are attached to a suspension matrix. These types of matrices canincrease localization efficiency. For example, a suspension matrixcomposed of DNA and an antibody can be used to specifically bind anantigen. For example, an antigen can be less than 1,000 kDa molecularweight and possibly too small for efficient localization on themicroporous material. By first interacting the antigen with an antibody,attached to a nucleic acid, for example, the antigen can be moreefficiently localized based on its association with the nucleic acid. Incertain aspects, reaction of the target molecule with a suspensionmatrix can substantially improve localization efficiency. Furtherdetails of the use of a suspension matrix to increase localizationefficiency are discussed below.

One way of categorizing analytes is by their size, relative to the poresize of the microporous material used to localize the analyte. Forexample, the analyte can have a contour length or globular diameter atleast 1.5 times, two times, three times, four times, six times, eighttimes, ten times, or twenty times the diameter of the pores in themicroporous material. It is understood that in certain embodiments thegoal is to localize the analyte near the surface of the microporousmaterial and then to detect the localized analyte. The larger theanalyte is relative to the pore size the more efficiently the analytewill be localized, meaning the fewer the number of analyte moleculesthat will escape from the localized position by passing through themicroporous material. However, it is understood that as the size of thepores are decreased, the speed at which the fluid is able to passthrough the filter or material is decreased, and the higher the numberof impurities that will be localized along with the desired analyte. Itis considered that analyte size can be determined and that calculationscan be used to determine the appropriate pore size of a material to beused, and that furthermore, empirical analysis can be used to determineor optimize the pore size as well.

i. Tissues

Samples can be obtained from any type of tissue of an organism. Forexample, in certain situations the tissue can be used directly, such aswhen the tissue is blood. However, in other situations, the tissue canbe collected and then manipulated, by for example, homogenization orgrinding etc.

There are four basic types of tissue. These compose all the organs,structures and other contents of an organism. There is the Epitheliumtissue, which lines, covers, protects, absorbs and secretes, within oron the organism. There is connective tissue which holds other tissuesand materials together. Blood is considered a connective tissue. Thereis muscle tissue, which contain muscle cells that contain contractilefilaments that move past each other and change the size of the cell.Lastly there is nervous tissue. Examples of tissue subtypes or organsthat contain more than one type of tissue would be, adipose, breast,brain, bone, intestine, stomach, skin, blood, liver, kidney, uterine,prostate, colon, urinary tract, cardiac, pulmonary, lung, muscle,ligaments, tendons, cartilage, semen, lymphatic, for example.

ii. Cells

Any type of cell can be considered an analyte. For example, eukaryoticcells and prokaryotic cells can be analytes. Examples of Eukaryoticcells that can be analytes are all types of animal cells, such as mammalcells, reptile cells, amphibian cells, and avian cells, blood cells,hepatic cells, kidney cells, skin cells, brain cells, bone cells, nervecells, immune cells, lymphatic cells, brain cells, plant cells, andfungal cells. In another aspect, the analyte can be a component of acell including, but not limited to, the nucleus, the nuclear membrane,leucoplasts, the microtrabecular lattice, endoplasmic reticulum,ribosomes, chromosomes, cell membrane, mitochondrion, nucleoli,lysosomes, the Golgi bodies, peroxisomes, or chloroplasts.

iii. Bacteria

Any type of bacteria can be an analyte. Examples of bacterium include,but are not limited to, Abiotrophia, Achromobacter, Acidaminococcus,Acidovorax, Acinetobacter, Actinobacillus, Actinobaculum, Actinomadura,Actinomyces, Aerococcus, Aeromonas, Afipia, Agrobacterium, Alcaligenes,Alloiococcus, Alteromonas, Amycolata, Amycolatopsis, Anaerobospirillum,Anaerorhabdus, Arachnia, Arcanobacterium, Arcobacter, Arthrobacter,Atopobium, Aureobacterium, Bacteroides, Balneatrix, Bartonella,Bergeyella, Bifidobacterium, Bilophila Branhamella, Borrelia,Bordetella, Brachyspira, Brevibacillus, Brevibacterium, Brevundimonas,Brucella, Burkholderia, Buttiauxella, Butyrivibrio, Calymmatobacterium,Campylobacter, Capnocytophaga, Cardiobacterium, Catonella, Cedecea,Cellulomonas, Centipeda, Chlamydia, Chlamydophila, Chromobacterium,Chyseobacterium, Chryseomonas, Citrobacter, Clostridium, Collinsella,Comamonas, Corynebacterium, Coxiella, Cryptobacterium, Delftia,Dermabacter, Dermatophilus, Desulfomonas, Desulfovibrio, Dialister,Dichelobacter, Dolosicoccus, Dolosigranulum, Edwardsiella, Eggerthella,Ehrlichia, Eikenella, Empedobacter, Enterobacter, Enterococcus, Erwinia,Erysipelothrix, Escherichia, Eubacterium, Ewingella, Exiguobacterium,Facklamia, Filifactor, Flavimonas, Flavobacterium, Francisella,Fusobacterium, Gardnerella, Gemella, Globicatella, Gordona, Haemophilus,Hafnia, Helicobacter, Helococcus, Holdemania Ignavigranum, Johnsonella,Kingella, Klebsiella, Kocuria, Koserella, Kurthia, Kytococcus,Lactobacillus, Lactococcus, Lautropia, Leclercia, Legionella,Leminorella, Leptospira, Leptotrichia, Leuconostoc, Listeria,Listonella, Megasphaera, Methylobacterium, Microbacterium, Micrococcus,Mitsuokella, Mobiluncus, Moellerella, Moraxella, Morganella,Mycobacterium, Mycoplasma, Myroides, Neisseria, Nocardia, Nocardiopsis,Ochrobactrum, Oeskovia, Oligella, Orientia, Paenibacillus, Pantoea,Parachlamydia, Pasteurella, Pediococcus, Peptococcus,Peptostreptococcus, Photobacterium, Photorhabdus, Plesiomonas,Porphyrimonas, Prevotella, Propionibacterium, Proteus, Providencia,Pseudomonas, Pseudonocardia, Pseudoramibacter, Psychrobacter, Rahnella,Ralstonia, Rhodococcus, Rickettsia Rochalimaea Roseomonas, Rothia,Ruminococcus, Salmonella, Selenomonas, Serpulina, Serratia, Shewenella,Shigella, Simkania, Slackia, Sphingobacterium, Sphingomonas, Spirillum,Staphylococcus, Stenotrophomonas, Stomatococcus, Streptobacillus,Streptococcus, Streptomyces, Succinivibrio, Sutterella, Suttonella,Tatumella, Tissierella, Trabulsiella, Treponema, Tropheryma,Tsakamurella, Turicella, Ureaplasma, Vagococcus, Veillonella, Vibrio,Weeksella, Wolinella, Xanthomonas, Xenorhabdus, Yersinia, and Yokenella.Other examples of bacterium include Mycobacterium tuberculosis, M.bovis, M. typhimurium, M. bovis strain BCG, BCG substrains, M. avium, M.intracellulare, M. africanum, M. kansasii, M. marinum, M. ulcerans, M.avium subspecies paratuberculosis, Staphylococcus aureus, Staphylococcusepidermidis, Staphylococcus equi, Streptococcus pyogenes, Streptococcusagalactiae, Listeria monocytogenes, Listeria ivanovii, Bacillusanthracis, B. subtilis, Nocardia asteroides, and other Nocardia species,Streptococcus viridans group, Peptococcus species, Peptostreptococcusspecies, Actinomyces israelii and other Actinomyces species, andPropionibacterium acnes, Clostridium tetani, Clostridium botulinum,other Clostridium species, Pseudomonas aeruginosa, other Pseudomonasspecies, Campylobacter species, Vibrio cholerae, Ehrlichia species,Actinobacillus pleuropneumoniae, Pasteurella haemolytica, Pasteurellamultocida, other Pasteurella species, Legionella pneumophila, otherLegionella species, Salmonella typhi, other Salmonella species, Shigellaspecies Brucella abortus, other Brucella species, Chlamydi trachomatis,Chlamydia psittaci, Coxiella burnetti, Escherichia coli, Neiserriameningitidis, Neiserria gonorrhea, Haemophilus influenzae, Haemophilusducreyi, other Hemophilus species, Yersinia pestis, Yersiniaenterolitica, other Yersinia species, Escherichia coli, E. hirae andother Escherichia species, as well as other Enterobacteria, Brucellaabortus and other Brucella species, Burkholderia cepacia, Burkholderiapseudomallei, Francisella tularensis, Bacteroides fragilis,Fudobascterium nucleatum, Provetella species, and Cowdria ruminantium,or any strain or variant thereof.

iv. Virons and viruses

Any type of virus can be an analyte. Examples of viruses include, butare not limited to, Herpes simplex virus type-1, Herpes simplex virustype-2, Cytomegalovirus, Epstein-Barr virus, Varicella-zoster virus,Human herpesvirus 6, Human herpesvirus 7, Human herpesvirus 8, Variolavirus, Vesicular stomatitis virus, Hepatitis A virus, Hepatitis B virus,Hepatitis C virus, Hepatitis D virus, Hepatitis E virus, Rhinovirus,Coronavirus, Influenza virus A, Influenza virus B, Measles virus,Polyomavirus, Human Papillomavirus, Respiratory syncytial virus,Adenovirus, Coxsackie virus, Dengue virus, Mumps virus, Poliovirus,Rabies virus, Rous sarcoma virus, Yellow fever virus, Ebola virus,Marburg virus, Lassa fever virus, Eastern Equine Encephalitis virus,Japanese Encephalitis virus, St. Louis Encephalitis virus, Murray Valleyfever virus, West Nile virus, Rift Valley fever virus, Rotavirus A,Rotavirus B, Rotavirus C, Sindbis virus, Simian Immunodeficiency cirus,Human T-cell Leukemia virus type-1, Hantavirus, Rubella virus, SimianImmunodeficiency virus, Human Immunodeficiency virus type-1, Vacciniavirus, SARS virus, and Human Immunodeficiency virus type-2, or anystrain or variant thereof.

v. Parasites

Any type of parasite can be an analyte. Examples of parasites include,but are not limited to, Toxoplasma gondii, Plasmodium falciparum,Plasmodium vivax, Plasmodium malariae, other Plasmodium species,Trypanosoma brucei, Trypanosoma cruzi, Leishmania major, otherLeishmania species, Schistosoma mansoni, other Schistosoma species, andEntamoeba histolytica, or any strain or variant thereof.

vi. Proteins

Any type of protein can be an analyte. For example, the protein caninclude peptides or fragments of proteins or peptides. The protein canbe of any length depending upon the pore size in the microporousmaterial, and can include one or more amino acids or variants thereof.The protein(s) can be fragmented, such as by protease digestion, priorto analysis. A protein sample to be analyzed can also be subjected tofractionation or separation to reduce the complexity of the samples.Fragmentation and fractionation can also be used together in the sameassay. Such fragmentation and fractionation can simplify and extend theanalysis of the proteins.

vii. Antibodies

Any type of antibody can be an analyte. As used herein, the term“antibody” encompasses, but is not limited to, whole immunoglobulin(i.e., an intact antibody) of any class, chimeric antibodies and hybridantibodies, with dual or multiple antigen or epitope specificities, andfragments, such as F(ab′)2, Fab′, Fab and the like, including hybridfragments. Thus, fragments of the antibodies that retain the ability tobind their specific antigens are provided. For example, fragments ofantibodies which maintain binding activity are included. Such antibodiesand fragments can be made by techniques known in the art. Methods forproducing antibodies and screening antibodies for specificity andactivity (See Harlow and Lane. Antibodies, A Laboratory Manual. ColdSpring Harbor Publications, New York, (1988)). The term “antibodies” isused herein in a broad sense and includes both polyclonal and monoclonalantibodies. In addition to intact immunoglobulin molecules, alsoincluded in the term “antibodies” are fragments or polymers of thoseimmunoglobulin molecules, and human or humanized versions ofimmunoglobulin molecules or fragments thereof. Also included within themeaning of “antibody” are conjugates of antibody fragments and antigenbinding proteins (single chain antibodies) as described, for example, inU.S. Pat. No. 4,704,692, the contents of which are hereby incorporatedby reference.

viii. Antigens

Any type of antigen can be an analyte. “Antigen” as used herein includessubstances that upon administration are capable of eliciting an immuneresponse, thereby stimulating the production and release of antibodiesthat bind specifically to the antigen. Antigens include molecules and/ormoieties that are bound specifically by an antibody to form anantigen/antibody complex. Examples of antigens include, but are notlimited to, peptides, polypeptides, proteins, nucleic acids, DNA, RNA,saccharides, combinations thereof, fractions thereof, or mimeticsthereof.

ix. Fungi

Any type of fungus can be an analyte. Examples of fungi include, but arenot limited to, Candida albicans, Cryptococcus neoformans, Histoplamacapsulatum, Aspergillus fumigatus, Coccidiodes immitis, Paracoccidiodesbrasiliensis, Blastomyces dermitidis, Pneomocystis carnii, Penicilliummarneffi, and Alternaria alternate, and variations or different strainsof these.

x. Effector Molecules

The analyte can be an effector molecule. “Effector molecules,” alsoreferred to as “effector species,” “effectors,” and “moleculareffectors,” are selected molecules capable of transforming energy intowork, work into energy, work or energy into information, or informationinto work or energy and include, but are not limited to,signal-generating species, stimulus-response molecules,response-generating molecules, enzymes, synthetic enzymes, drugs,catalytic antibodies, catalysts, contractile proteins, transportproteins, regulatory proteins, redox proteins, redox enzymes, redoxmediators, cytochromes, electroactive compounds, photoactive compounds,supermolecules, supramolecular devices and shape-memory structures, orany derivative or variant thereof.

xi. Ligands

Any type of ligand can be an analyte. The term “ligand” is a moleculecapable of specifically binding to a receptor by affinity-basedinteraction. Ligands include, but are not limited to, receptor agonists,partial agonists, mixed agonists, antagonists, response-inducing orstimulus molecules, drugs, hormones, pheromones, transmitters,autacoids, growth factors, cytokines, prosthetic groups, coenzymes,cofactors, substrates, precursors, vitamins, toxins, regulatory factors,antigens, haptens, carbohydrates, molecular mimics, print molecules,structural molecules, effector molecules, selectable molecules, biotin,digoxigenin, and congeners, crossreactants, analogs, competitors orderivatives of these molecules as well as library-selected moleculescapable of specifically binding to selected targets and conjugatesformed by attaching any of these molecules to a second molecule, or anyderivative or variant thereof.

xii. Receptors

The analyte can be a receptor. The term “receptor” is a molecule capableof specifically binding to a ligand by affinity-based interactions.“Receptors” include, but are not limited to, biological, synthetic orengineered membrane receptors, hormone receptors, drug receptors,transmitter receptors, autacoid receptors, pheromone receptors,stimulus-response coupling or receptive molecules, antibodies, antibodyfragments, engineered antibodies, antibody mimics or mimetics, molecularmimics, molecular imprints, molecular recognition units, adhesionmolecules, agglutinins, lectins, selecting, cellular receptors, avidinand streptavidin, and congeners, analogs, competitors or derivatives ofthese molecules as well as nonoligonucleotide molecules selected, e.g.,by combinatorial methods and/or library screening, to specifically bindother selected molecules and conjugates formed by attaching any of thesemolecules to a second molecule. Receptors further include selectedmolecules capable of specifically recognizing structural molecules,effector molecules and selectable molecules comprising ligands, or anyderivative or variant thereof.

xiii. Signal-Generating Molecules

The analyte can be a signal generating molecule. “Signal-generatingmolecules” and “signal-generating species” are molecules capable ofgenerating a detectable signal or enhancing or modulating thedetectability of a substance or transducing an energy, activity, outputor signal of a substance into a qualitatively, quantitatively ordetectably different energy, activity, output, signal, state or form.Alternatively, signal-generating molecules can interact with the targetmolecule to produce an analyte capable of localization.Signal-generating molecules include, but are not limited to, molecules,groups of molecules, conjugates and complexes comprising detectable (andoptionally dyed, modified, conjugated, labeled or derivatized) tags,tracers, radioisotopes, labels, reporters, polymers, light-harvestingcomplexes, antenna structures, natural and synthetic and biomimeticphotosynthetic molecules, reaction centers, photosystems, signaltransduction pathways, molecular cascades, macromolecules,microparticles, nanoparticles, colloids, metals, dyes, fluorophores,phosphors and other photon-absorbing, photon-emitting and photosensitivemolecules, including molecules or groups that enhance, attenuate,modulate or quench the photon-absorbing or photon-emitting properties ofanother molecule or group, energy transfer donors and acceptors,enzymes, coenzymes, cofactors, catalytic antibodies, synthetic enzymesand catalysts, molecular mimics and mimetics, luminescent,triboluminescent, sonoluminescent, electroluminescent, chemiluminescentand bioluminescent molecules, electron transfer donors and acceptors,oxidizing and reducing compounds, mediators and other electroactivemolecules, metabolic, photoactive, signaling and signal processingmolecules used to capture and transduce energy in biological andbiomimetic processes and systems, optionally including natural,synthetic or mimetic scaffold, organizational and coupling molecules,chaperones and accessory biological or biomimetic molecules or groups ofmolecules involved in the transduction of a first form of energy orinformation into a second form of energy or information.

xiv. Structural Molecules

The analyte can be any structural molecule. Examples include, but arenot limited to, elements, atoms, molecules, ions, and compoundscomprising surfaces, amphibious surfaces, inorganic and organicmaterials such as carbon, silicon, glass, organic and inorganiccrystals, selected solvents, selected solutes, natural, biomimetic andsynthetic nanostructures and microstructures, fibers, filaments, silks,molecular scaffolds, nanotubes, nanorods, fullerenes, buckyballs,diamondoid molecules, semiconductors, insulators, metals, plastics,elastomers, polymers, detergents, lubricants, waxes, oils, powders,fillers, excipients, fibers, tableting ingredients, packaging materials,papers, industrial plastics, cyclic and polycyclic molecules, dendrons,dendrimers, electrolytes and polyelectrolytes, salts, hydrocarbons,ceramics and biological, biocompatible, biomimetic, biodegradable andimprintable monomers, multimers and polymers, e.g., fatty acids, lipids,surfactants, amino acids, peptides, proteins, polyamines, polyacids,sugars, starches, cellulose, glycosylated molecules, glycopolymers andconjugates thereof.

xv. Ions

The analyte can be any ion. Examples of ions include alkali metal ions,alkali earth metal ions, transition metal ions, or lanthanide metalions. In one aspect, a chelate can be added to a sample containing theion to form an ion/chelate complex prior to localization. In thisaspect, the ion, the chelate, and/or the ion/chelate complex is theanalyte. In another aspect, a chelate can be covalently attached nearthe surface of the microporous material that can interact with the ion.

xvi. Nucleic Acid Analytes

In one aspect, the analyte is a nucleic acid. Nucleic acids such asdeoxyribonucleic acid (DNA), ribonucleic acid (RNA), and peptide nucleicacid (PNA) are polymeric, polyionic molecules soluble in aqueoussolution under certain conditions. The assumed three-dimensionalstructures of nucleic acids in solution as a function of pH, ionicstrength, counter ions, charge neutralization, hydration, organicprecipitants, molecular composition, etc., are known by those skilled inthe art. In one aspect, the nucleic acid can be single or doublestranded DNA or RNA.

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids as well as any otherproteins disclosed herein, as well as various functional nucleic acids.The disclosed nucleic acids are made up of for example, nucleotides,nucleotide analogs, or nucleotide substitutes. Non-limiting examples ofthese and other molecules are discussed herein. It is understood thatfor example, when a vector is expressed in a cell, the expressed mRNAwill typically be made up of A, C, G, and U.

xvii. Nucleotides and Related Molecules

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Annon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to nucleotides are well known in the art and would includefor example, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, and 2-aminoadenine as well as modifications atthe sugar or phosphate moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety. (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86, 6553-6556),

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH2 or O) at the C6 position of purinenucleotides.

Thus, nucleic acids are polymers made up of nucleotides, called basesgenerically. The nucleic acid molecules can be characterized by thenumber of bases that make up the nucleic acid. For example, in certainembodiments the nucleic acid analytes are at least 100, 101, 102, 103,104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117,118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131,132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,146, 147, 148, 149, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195,200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 295, 300, 320, 340,360, 380, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900,2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3200,3400, 3600, 3800, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000,8500, 9000, 9500, 10000, 11000, 12000, 13000, 14000, 15000, 16000,17000, 18000, 19000, 20000, 25000, 50000, 100000, 200000, 300000,400000, 500000, and 1000000 bases or base pairs long. In another aspect,the DNA or RNA has at least about 1,500 bases or base pairs.

The primary structure of nucleic acids is linear, with each base or basepair contributing approximately 0.34 nm to the fully extended contourlength. Representative contour lengths of various nucleic acids areshown in Table 1.

TABLE 1 Nucleic Acid Bases or Base Pairs Contour Length Oligo Probe 40.013 microns 200 .068 microns 1,000 .340 microns HIV RNA 9,000 3.06microns T4 Phage genome 160,000 54.4 microns E. Coli genome 4.2 million1,428 microns 

The assumed diameter of nucleic acid molecules is about 2 nm, resultingin an exceptionally anisotropic, high aspect ratio primary structure.Higher order structures (secondary, tertiary, etc.) are well known fornucleic acids and are highly environment dependent. In aqueous solution,free of organic, ionic, or polymeric precipitants, nucleic acids areusually described as a coiled or relaxed three-dimensionalconfiguration, but may be reversibly precipitated with known agents intoa globular or condensed state. Likewise, coil form nucleic acids areknown to undergo substantial three dimensional changes such aselongation, molecular orientation, etc. in flowing solutions.

In one aspect, the microporous materials, methods, and articlesdescribed herein can isolate nucleic acids from biological fluids fordiagnostic purposes. High sensitivity assays require a 200 μl or largerpatient sample to capture sufficient nucleic acid to be statisticallymeaningful. Commercially available viral load assays employ target orsignal multiplication techniques (e.g., PCR, βDNA, etc.) based on targetviral nucleic acid. That is, these techniques quantify total viralnucleic acid present in the sample fluid after virion lysis. A “virion”is a complete, mature virus particle. Unencapsulated viral nucleic acidspresent in the sample fluid before lysis are also included as part ofthe total load. Since unencapsulated viral nucleic acids are widelyregarded as non-infectious, their inclusion into the viral load totalrepresents an inaccuracy in the measurement.

Described herein are methods and materials that can be used to improveexisting viral load assays by removing the majority of unencapsulatednucleic acids prior to analysis. In one example, the virion assayed isan HIV virion. The patient sample (e.g., plasma, serum, CSF, biologicalwarfare extracts, etc.) is filtered through a microporous materialdescribed herein. The microporous material localizes all soluble nucleicacids (e.g., ssDNA, dsDNA, ssRNA, dsRNA) as well as cellular componentslarger than several microns in diameter. Intact virions pass through thefilter, and the patient sample filtrate is then lysed to free the viralnucleic acids. The lysed patient sample filtrate is filtered then rinsedthrough a second microporous material to localize the virion-derivednucleic acids near the surface of the second microporous material.

3. Filters

Filtration can be considered a separation process based primarily onmolecular dimension in relation to flow path dimension of the filter.Filters are generally classified as surface filters, where filtrationprimarily occurs on the entrance surface of the filter due to small,uniform pore dimensions of the microporous material, or depth filters,where the pores of the microporous material have a tortuous pathstructure and entrap particles within the depth of the filter.

Any of the microporous materials described herein, including thecomposites and modified-microporous materials, can be manufactured as afilter. In one aspect, the microporous material can be formulated into afilter such as, for example, a bead or membrane. Beads can be fromseveral millimeters in diameter down to less than one micron diameterdepending on system requirements. Beads can be spherical, irregularlyshaped, or formed into any shape as required. The size of the beads canalso vary depending upon their application. Beads can be uniformlyporous, or display a microporous surface overlying a limited porositycore. Additionally, beads can be magnetic, paramagnetic, or have otherproperties that aid in bead separation.

Membranes are two dimensional structures with limited thickness. In oneaspect, membrane filters have a thicknesses of from less than 1 micronto greater than 100 microns. In one aspect, the membrane has a thicknessof 1 micron to 100 microns, 10 microns to 90 microns, 20 microns to 80microns, 30 microns to 70 microns, or 40 microns to 60 microns. Inanother aspect, 0.2 micron Anopore™ inorganic oxide membranes aretypically 40 to 50 microns thick, while 0.02 micron Anopore membraneshave a very thin 0.02 micron porous layer less than 1 micron thickoverlying a 0.2 micron support layer that is approximately 40 micronsthick. Accordingly, membranes may be formed or created as an analytelocalization surface on a support material. This support materialconfers useful characteristics to the system, such as strength, ease ofhandling, fluid flow properties, thermal properties, etc. In thisaspect, the support material, and therefore the membrane, may be ofvarying shape, size, and design consistent with overall requirements.

In the methods described herein, surface filters or depth filters can beused for prefiltration of impurities and other debris from a samplebefore analyte separation and localization. To assist with improvingfilterability of test samples, other techniques known by those skilledin the art can be used in combination with any of the articles describedherein. These include prefiltering test samples to remove largeimpurities, or treatment of test samples such as by sample digestioninvolving the use of enzymes, use of surfactants, use of chaotropicagents, ultrasonic and thermal treatments, protein precipitation, andthe like.

4. Surfaces

The microporous material possesses many different surfaces. For example,there are planar surfaces, external surfaces, internal surfaces, andwetted surfaces. A schematic of a filter composed of a microporousmaterial is depicted in FIGS. 1 a and 1 b. FIG. 1 a is a top-view of thefilter showing a microporous material 100, where 110 represents one ofmany holes or pores in the microporous material. These holes do not needto be uniformly spaced or sized, but in many embodiments they are. Thereare many different surfaces associated with the filter. FIG. 1 b is across-sectional view of the filter with a microporous material. Forexample, there is a planar surface 120, which is the surface of themicroporous material defined by the plane produced by the externalsurface of the microporous material 130 and the openings 110 caused bythe plurality of pores and holes. An internal surface 140 is formed bythe channel 150 that runs through the microporous material.

5. Analyte Interaction with Microporous Material

i. Localized

When the analyte is contacted with any of the microporous materialsdescribed herein, the microporous material prevents or inhibits theanalyte from passing into and through the material. The microporousmaterial interacts with the analyte to substantially prevent molecularcollapse of the analyte through the microporous material. In one aspect,surface filtration can be used to rapidly localize and concentrate theanalyte. The efficiency of the surface filtration will depend uponnumerous parameters such as analyte to pore size ratio, composition ofimpurities in the fluid to be filtered, and analyte-to-surfaceinteractions.

In the aspect described above, when the sample containing the analytecomes into contact with the microporous material, the analyte islocalized on the microporous material. The term “localized” as usedherein is defined as an increase in the concentration of the analytenear the surface of the material. Thus there is reduction or inhibitionof the analyte from flowing into and/or through the microporousmaterial.

The localized analyte is near the surface of the microporous materialand not trapped within the microporous material. Referring to FIG. 1 b,the term “near the surface” as used herein is defined as (1) the region10 microns below the planar surface of the microporous material, whichis 160, (2) adjacent to the planar surface 120 of the microporousmaterial, or (3) 25 μm above the planar surface, which is 170. In oneaspect, the entire analyte or a portion of the analyte is at most 10microns below the planar surface of the microporous material. In oneaspect, when the analyte is adjacent to the planar surface, the analyteis in direct contact with the external surface of the microporousmaterial. In one aspect, the analyte is below the planar surface by 10%relative to the pore depth, 8% relative to the pore depth, 6% relativeto the pore depth, 4% relative to the pore depth, 3% relative to thepore depth, or 2% relative to the pore depth In one aspect, the analyteis from 10 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm, or 0.5 μm below the planarsurface of the microporous material to 25 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6μm, 5 μm, 4 μm, 3 μm, 2 μm, or 1 μm above the planar surface, where anyendpoint below the planar surface can be used with any point above theplanar surface.

In one aspect, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%of the analyte is localized near the surface of the microporousmaterial. In another aspect, the amount of analyte that is localized isat least 95%, at least 96%, at least 97%, at least 98%, at least 99%, atleast 99.5%, or 100%. In one aspect, the target analytes areconcentrated near the surface of the microporous material as discrete,isolated molecules. This aspect is useful for molecular counting, whichis discussed below. In another aspect, the target molecules can belayered on top of each other near the surface of the microporousmaterial. Localization of the analyte by mechanical filtration providesan extremely rapid, uncomplicated method of obtaining pure analytes forsubsequent processing.

In addition, unlike prior techniques that require diffusion controlledreactions to retain the analyte for molecular counting, the microporousmaterials described herein use weak surface interaction to localize theanalyte near the surface of the material. For example, when the analyteis a nucleic acid, reversible nucleic acid processing, as well asstronger surface interaction such as hybridization, covalent attachment,etc. for rapid nucleic acid localization to a filter surface arepossible when using any of the microporous materials described herein.Although optional, a binding oligonucleotide that is complimentary tothe nucleic acid that is to be localized does not need to be present onthe microporous material in order to localize the nucleic acid. Inanother aspect, for ultra low concentration nucleic acid detection, suchas found in ultra sensitive viral load assays, the microporous materialscan be used with optical molecular counting techniques to directlydetermine nucleic acid concentrations without molecular amplification.This is possible because of the high concentration effect andexceptional detectability of nucleic acids localized near the surface ofthe microporous material.

As described above, separation of the nucleic acid is facilitated byweak nucleic acid-to-surface interactions near the surface of themicroporous material. Although considered rigid on a molecular scale,nucleic acids are deformable on a micron size scale and, under certainconditions, can pass through practical filter pore diameters. Separationof nucleic acids by localization involves an interaction between theanalyte and the microporous material in order to prevent the analytefrom collapsing into the microporous material. In view of this, the sizeof the pores present in the microporous material is one variable toconsider when selecting the microporous material. The pores should besmall enough to efficiently exclude the analyte, but large enough topermit rapid passage of impurities remaining in the sample fluid. Aschematic depiction of the technique is illustrated in FIG. 2.

As shown in FIGS. 2 a and 2 b, the analyte 200 has been localized nearthe surface of the microporous material 210, having a plurality ofmicropores 220 and flats 230, by mechanical filtration from a flowingsolution 240. The length of the analyte 200 is large compared to thedimensions of micropores 220 and physically spans several openings asdepicted in FIG. 2 a.

As shown in FIG. 2 b, surface interactions occur between localizedanalyte 200 and the exposed surface on flats 230. The interactionsbetween polymers and surfaces have been widely studied and vary fromessentially zero interaction to complete covalent immobilization. Byanalogy to purely mechanical systems, if solution 240 in FIG. 2 b isflowing through the filter, analyte 200 is supported across micropores220 by a molecular tension 250, which is supported by shear forces 260developed between analyte 200 and flats 230. This is a consequence ofthe relatively large pore diameter preferred for practical filtration inconjunction with known molecular deformability. Even for large analytesspanning many pores, shear forces 260 are required to prevent thecollapse of analyte 200 through micropores 220.

Unlike previously known analyte purification methodologies that rely onadsorption/precipitation within the pores of silica or other poroussupports, when the analyte is a nucleic acid, the weak interactionsrequired for nucleic acid localization is achievable with soluble, coilform nucleic acids under mild conditions. In one aspect, weak surfaceinteraction for reversible nucleic acid processing is contemplated. Inanother aspect, stronger surface interactions such as hybridization,covalent attachment, etc. can be used for rapid nucleic acidlocalization.

ii. Immobilized

The term “localized” also includes the immobilization of an analyte nearthe surface of the microporous material. The term “immobilized” as usedherein is defined as the restraint of the analyte at a specific regionnear the surface of the microporous material for a time long enough tobe counted. In one aspect, the analyte is immobilized in a 1 μm³ regionduring a specific detection period. The term “localized” also includesconcentrating the analyte near the surface of the microporous material.The phrase “concentrating the analyte” as used herein is defined as theaccumulation of the analyte near the surface of the microporous materialat a fixed area regardless of the purity of the concentrated analyte.When the analyte is localized near the surface of the microporousmaterial, the analyte may be reversibly or irreversibly bound to themicroporous material. When the analyte is “reversibly” bound, themajority of the analyte can be removed from the microporous material.Conversely, when the analyte is “irreversibly” bound, the majority ofthe analyte cannot be removed from the microporous material.

6. Conditions for Localization

In one aspect, in order to assist with analyte localization, variousinorganic salts can optionally be utilized in the sample solutions. Forexample, salts such as NaCl or chaotropic salts including guanidiniumisothiocyanate, guanidinium hydrochloride, and the like, can also beutilized as desired in the sample solutions. In one aspect, the amountof salt used is less than 2 M, such as 1.8 M, 1.6 M, 1.4 M, 1.2 M, 1.0M, 0.8 M, 0.6 M, 0.4 M, 0.2 M, 0.1 M, 0.05 M, where any concentrationcan form a range with another concentration (e.g., 0.1 M to 1.8M).

In another aspect, buffers can also be added to the sample containingthe analyte, in order to facilitate localization. In one aspect, tris,carbonate/bicarbonate and the like can be used. In another aspect,phosphate buffers can be used to reverse the retention of the analyte onthe microporous material and, thus, the analyte can be easily removedfrom the microporous material for subsequent analysis when desired.Phosphate ions are known to bind to aluminum oxide chromatographypacking and can compete for the same weak binding sites on themicroporous material. In one aspect, buffers can be used to controlsample pH. In one aspect, pH values lower than about 4 result in highnucleic acid localization efficiency independent of buffering ions. Inanother aspect, pH values greater than 9 to 10 show decreasinglocalization efficiency even with amine modified microporous surfaces.In one aspect, the sample pH is maintained in the range of 6 to 8 tominimize protein precipitation. In this pH range, the ability ofmicroporous material to efficiently localize nucleic acids depends onsurface modification, salt concentration, and ion additives, such asbuffer salts. In another aspect, an enzyme or surfactant can be presentin the sample. As more fully discussed later, the effect of variousbuffers, salts, proteins, surfactants, etc. on localization depends onthe nature of the interaction between the microporous surface and theanalyte. For example, localization of nucleic acids onto plain,unmodified inorganic metal oxide microporous surfaces of 0.2 micronnominal pore size, for instance Anopore 0.2 micron membrane filters,shows increasing localization efficiency with salt concentration. Asdescribed herein, this salt effect does not depend on whether the saltis chaotropic. Additionally, this salt effect indicates only lowconcentrations of salt are required to produce efficient localization,generally under 1 molar and frequently under 100 millimolar. Aminemodified Anopore 0.2 micron membrane filters show efficient localizationindependent of salt concentration.

Materials that coat microporous surfaces, for instance, proteins,surfactants, and the like, can affect localization by modification ofthe analyte-surface interaction. For example, proteins and surfactantsgenerally lower localization efficiency for unmodified 0.2 micronAnopore membrane filters, but have little effect on amine modifiedfilters. As previously mentioned, certain ions, for instance phosphate,borate, bicarbonate, etc. can affect nucleic acid localization on plain,unmodified 0.2 micron Anopore membrane filters presumably by competitionwith the analyte for weak binding sites on the microporous surface. Inone aspect, Amine modified 0.2 micron Anopore membrane filters showgreatly reduced ion effects.

7. Microporous Material

The term “microporous material” as used herein is any material having aplurality of pores, holes, and/or channels. The microporous materialpermits the flow of liquid through or into the material. The microporousmaterial generally possesses a high concentration of small, uniformholes or pores of sub-micron dimensions. The microporous material can beoptically transmissive to visible light, ultraviolet light, or infraredlight, depending on the particular detection technique used to analyzethe localized analyte. The microporous material can be hydrophilic topermit the rapid flow of water through the material. The microporousmaterial can be optically opaque if desired. It is also desirable thatthe microporous material also possess good mechanical strength for easyhandling, low non-specific binding, and relative inertness with theanalyte. The term “microporous material” as used herein includes any ofthe composites and modified-microporous materials discussed below.

In one aspect, the micropores can have diameters ranging in size fromabout 0.02 microns to about 0.2 microns. For very large analytes, suchas intact bacteria, cells, particulate, etc. larger pore sizes arecontemplated. As previously discussed, larger pore sizes are less proneto plugging with impurities generally found in some samples. Pore sizesup to 10 microns can be contemplated for analysis of certain samples. Inanother aspect, the diameter of the pores is from about 0.02 microns toabout 0.18 microns, about 0.02 microns to about 0.16 microns, about 0.02microns to about 0.14 microns, about 0.02 microns to about 0.12 microns,about 0.02 microns to about 0.1 microns, about 0.04 microns to about 0.2microns, about 0.06 microns to about 0.2 microns, about 0.08 microns toabout 0.2 microns, or about 0.1 microns to about 0.2 microns.

The microporous material can be composed of any material that has a highconcentration of small, uniform holes or pores or that can be convertedto such a material. Examples of such materials include, but are notlimited to, inorganic materials, polymers, and the like. In one aspect,the microporous material is a ceramic, a metal, carbon, glass, a metaloxide, or a combination thereof. In another aspect, the microporousmaterial includes a track etch material, an inorganic electrochemicallyformed material, and the like. The phrase “inorganic electrochemicallyformed material” is defined herein as a material that is formed by theelectroconversion of a metal to a metal oxide. The phrase “track etchmaterial” is defined herein as a material that is formed with the use ofionizing radiation on a polymer membrane to produce holes in thematerial. Such materials are commercially available. Any of themicroporous materials disclosed in U.S. Pat. No. 5,716,526, which isincorporated by reference in its entirety, can be used. In a furtheraspect, when the microporous material is a metal oxide, the metal oxideincludes aluminum oxide, zirconium oxide, titanium oxide, a zeolite, ora combination thereof. Examples of zeolites include, but are not limitedto, those disclosed in U.S. Pat. Nos. 4,304,675; 4,437,429; 4,793,833;and 6,284,232, which are incorporated by reference in their entireties.In one aspect, when the microporous material is a metal oxide, the metaloxide can contain one or more metal salts in varying amounts. Forexample, aluminum salts such as aluminum phosphate, aluminum chloride,or aluminum sulfate can be part of the microporous material.

In one aspect, the microporous material is an inorganic electroformedmetal oxide, such as described in U.S. Pat. No. 6,225,131, thedisclosure of which is incorporated by reference herein. Such ceramicmembranes are available from Whatman, Inc. and distributed under thetrade names Anopore™ and Anodisc™. Anopore membranes have a honeycombtype structure with each pore approximately 0.2 micron in diameter by 50microns long. The Anopore membranes are composed of predominantlyaluminum oxide with a small amount (5-10%) of aluminum phosphate. Inthis aspect, these microporous materials have the following desirablecharacteristics: substantially nonfluorescent in the visible spectrum;very uniform pore structure; high pore density and high liquid flowrates; relatively rigid; flat; and optically clear when wet; highmelting point and easily heat welded to plastic; relatively biologicallyinert, and little non-specific binding or denaturing effects forproteins, nucleic acids, etc. In one aspect, the microporous materialcan be aluminum or titanium that has been anodized. Anodization is atechnique known in the art that is used to produce an oxide layer on thesurface of the aluminum or titanium.

In another aspect, the microporous material can be chemically modifiedto enhance surface localization of analytes. For example, since nucleicacids are negatively charged molecules, the microporous material can betreated to have a positive charge with various chemicals so that thenucleic acids stick near the surface of the microporous material throughionic attractive forces. Such weak attractive forces aid in keeping thenucleic acids from passing through the microporous material. In oneaspect, the microporous material can be pretreated with silanizationreagents including, but not limited to, aminopropyltrimethoxysilane(APS), ethylenediaminopropyltrimethoxysilane (EDAPS), or other aminosilane reagents to impart a slight positive surface charge. In anotheraspect, the microporous material can be pretreated with polymermaterials, including but not limited to polylysine, to impart a slightsurface charge to enhance analyte localization. Additionally, themicroporous material can be modified with neutral reagents such as adiol, an example of which is acid hydrolyzedglycidoxypropyltrimethoxysilane (GOPS), to vary analyte retention.

Any of the microporous materials described herein can be modified bytechniques known to those skilled in the art. In one aspect, EDAPSmodified microporous materials can be prepared by dissolving about 5%EDAPS into molecular grade water at room temperature. The aqueoussolution is incubated for about 5 minutes at room temperature tohydrolyze the trimethoxy groups. This solution is filtered through themicroporous material at a rate of approximately 1 milliliter per 1 cm²over a period of about 5 minutes. The activated microporous material isrinsed by filtering several equivalent volumes of molecular grade waterthrough the microporous material, and is then dehydrated in a vacuumoven at approximately 40° C. The dry microporous material is then rinsedby filtering several equivalent volumes of 1×TE buffer solution (10 mMtris-HCl (tris(hydroxymethyl)aminomethane base), 1 mM EDTA(ethylenediaminetetraacetic acid) buffer, etc. pH 7-8) to adjust surfacepH. The microporous material is then rinsed by filtering severalequivalent volumes of molecular grade water and is then againdehydrated. The dry filter is stored dry at room temperature. In anotheraspect, diol modified membrane filters can be made similarly using GOPSinstead of EDAPS and including a well known acid hydrolysis step toconvert the adsorbed epoxide to a diol.

8. Composites

A few commercially available microporous materials possess lowautofluorescence, such as, for example, Anopore inorganic oxidemembranes. However, in some applications, even small amounts ofautofluorescence are detrimental. For instance, fluorescence microscopyis a widely used powerful analytical tool for biological andmorphological analysis. Weakly fluorescent specimens must be prepared onsurfaces with exceptionally low autofluorescence such as speciallyprocessed fused silica, glass, or crystalline silicon. Fluorescentanalysis of small particles (e.g., cells, virions, nucleic acids, etc.)directly on a filtration surface is typically limited by filtrationmembrane autofluorescence. Although inorganic oxide microporousmaterials exhibit much lower autofluorescence than polymeric membranes,the autofluorescence may be too high for analysis of weakly fluorescingparticles.

As described above, Anopore membranes have a honeycomb type structure,with each pore approximately 0.2 micron in diameter by 50 microns long.The internal area of the microporous material is much greater than theexternal surface area. Indeed, it is generally accepted the internalwetted area of Anopore membranes is typically 500 times greater than theprojected surface area. In some cases, this large internal surface areais detrimental to optical analysis of particles present on the externalsurface. For instance, biological cells are frequently analyzed byfluorescently tagged specific protein binding reactions. In theory, asystem can be developed that localizes unknown cells near the surface ofthe microporous material followed by exposing the localized cells tofluorescently labeled proteins. After rinsing unbound proteins from themicroporous material, specific binding reactions between the localizedcells and the labeled proteins are detected by increase in fluorescencein the localized cells. Unfortunately, all surfaces exhibit what isknown as nonspecific binding (NSB). That is to say, a small amount oflabeled protein will bind to the microporous material in the absence ofa specific receptor on a cell. Most optical detectors are unable todifferentiate specific signals originating from the specifically labeledsurface localized cells from that originating within the transparentmicroporous material from fluorescent proteins nonspecifically bound tothe large internal membrane surface area. Although the microporousmaterials described above are capable of effectively concentrating andlocalizing particulate samples to their surface for analysis, NSB to thelarge internal surface area of the microporous material frequentlylimits useful sensitivity.

In order to address these concerns, described herein are compositescomposed of microporous materials that have been modified to alter theoptical properties of the microporous material (e.g., autofluorescence,internal optical scatter, and fluorescent NSB detection). In one aspect,the microporous material is modified to render the microporous materialoptically opaque so as to reduce detectable fluorescence and scatteroriginating within the material, either from the material itself(autofluorescence) or from internally bound tracers or contaminants(NSB).

i. Pigment Containing Composites

The term “composite” as used herein is a product composed of two or morematerials. Depending upon the selection of the materials, the materialsmay or may not react with one another to produce the composite. In oneaspect, the composite is composed of a microporous material and apigment. The term “pigment” as used herein is any compound that modifiesthe optical properties of the microporous material. The term “pigment”also includes precursor compounds that upon chemical manipulation can beconverted to the pigment. For example, PdCl₂ may not be able to modifyoptical properties; however, when it is reduced to palladium metal, itis material than can modify optical properties of a microporousmaterial. The pigment is incorporated in the microporous material. Thephrase “incorporated in the microporous material” as used herein isdefined as any method for attaching a pigment to the microporousmaterial. The attachment of the pigment to the microporous material canbe via a covalent bond, an ionic interaction, entrapment of the pigmentby the pores of the microporous material, or by depositing the pigmentnear the surface of the microporous material and/or on the internalsurface of the microporous material. The pigment can be incorporated onany wettable surface of the microporous material. Any of the compositesdescribed herein can be used in any of the articles, methods, or kitsdescribed herein.

The composite is formed by incorporating a pigment in the microporousmaterial. In one aspect, the pigment is covalently attached to themicroporous material. The phrase “covalently attached to the microporousmaterial” is defined herein as forming a chemical bond directly betweenthe pigment and the microporous material in the absence of a linker. Forexample, a moiety on the surface of the microporous material can reactwith a moiety present on the pigment to form a covalent bond. Thepigment can be covalently attached to any wettable surface of themicroporous material (i.e., internal and external surfaces). Examples ofmoieties bound to the microporous material that can react with thepigment include, but are not limited to, a hydroxyl group, a carboxylgroup, a sulfhydryl group, an amine group, or a combination thereof.

Alternatively, the phrase “covalently attached” also includes attachingthe pigment to the surface of the microporous material with the use of alinker. For example, a linker can be any compound having a moiety thatis capable of forming a bond with a moiety on the surface of themicroporous material as well as a moiety that is capable of forming acovalent bond with the pigment. The moiety on the linker can be the sameor different depending upon the microporous material and pigmentselected. In one aspect, the linker is an organosilyl group. Silanationhas been previously described and is a well-understood chemical methodof modifying the surface of materials and can be additionally used tocreate a reactive surface on which to attach various organic molecules.For example, amine terminated reactive silanation reagents such asaminopropyl trimethoxysilane (APS) and ethylenediaminopropyltrimethoxysilane (EDAPS) can be used to create a surface capable ofcovalent reaction with protein linkers, reactive dyes, etc.Additionally, epoxy terminated reactive silanation reagents such asglycidoxypropyl trimethoxysilane (GOPS) can be used to create a surfacecapable of directly reacting with nucleophiles in dyes, proteins, orother reagents. In one aspect, the microporous material can be modifiedby those skilled in the art with other silanation reagents to impartvarious useful surface functionalities, such as carboxylate, sulfhydryl,hydroxy, aromatic, hydrophobic. Any of the silane compounds disclosed inU.S. Pat. No. 5,959,014, which is incorporated by reference in itsentirety, can be used in this aspect.

In one aspect, a pigment in the form of an organic dye is covalentlyattached to the wettable surface of the microporous material. The term“organic dye” as used herein is defined as any organic compound that canmodify the optical properties of the microporous material when thecompound is incorporated in the microporous material. Dye attachment tosilane modified microporous material is straightforward and depends onthe specific attachment chemistry chosen. For instance, amino modifiedmicroporous material (e.g., APS or EDAPS) can react with severalchemical families of known amino reactive dyes including, but notlimited to, isothiocyanates, triazines, and active esters. Any of theorganic dyes disclosed in U.S. Pat. Nos. 6,630,018; 6,623,908;5,985,514; and 5,294,870, which are incorporated by reference in theirentireties, can be used to produce the composites described herein.

In another aspect, the organic dye can stain the microporous materialwithout forming a covalent bond with the microporous material. Forexample, the organic dye can be entrapped in the pores of themicroporous material. Additionally, the organic dye can be attached to asurface bound polymer such as, for instance, polylysine.

The organic dye that is selected depends on the final desired propertiesof the membrane. For example, triazinyl dyes are widely used aspermanent fiber reactive dyes for cloth, paper, etc. as well asanalytical labeling. They are available both as fluorescent andnon-fluorescent dyes. Dichlorotriazinylaminofluorescein (DTAF) can reactwith amine modified microporous materials to produce an intenselyfluorescent yellow material. Low fluorescent triazinyl dyes, such asReactive Blue 4 or selections from the Procion MX series of organic dyescan be used to impart reduced autofluorescence, NSB fluorescence, andmicroporous material scatter.

In one aspect, the composite includes a microporous material composed ofaluminum oxide and the pigment is an organic dye, wherein the organicdye is covalently attached to the aluminum oxide by an organosilylgroup. In another aspect, the microporous material is Anopore.

In another aspect, the pigment is deposited on the microporous material.The term “deposited on the microporous material” is defined herein asany method for precipitating the pigment on any wettable surface of themicroporous material. In one aspect, the resultant precipitate ordeposit can be composed of ionic compounds, non-ionic compounds,elemental compounds, or a combination thereof. In one aspect, thepigment is an elemental metal such as a transition metal, a metal oxide,a metal alloy, or a combination thereof. Examples of transition metalsinclude palladium, nickel, silver, gold, or a combination thereof. Inthe case when the pigment is a transition metal, the metal can bezero-valent or any other valency depending upon the particulartransition metal compound selected. In another aspect, carbon blackinclusions can be deposited in the pores of the microporous material.

In one aspect, the pigment can be deposited on the microporous materialby an electroless metallization process. Electroless metallizationprocesses are known to those skilled in the art for depositing metalalloys within high aspect ratio nanometer scale pore type structures.Electroless metallization processes on non-conductors frequently requirea surface catalyst for initiation. In one aspect, palladium metal can beused in several forms, including colloid suspensions, ionic liquids, andmetal ions that may be chemically reduced on the surface of themicroporous material to form metallic nanoscale clusters. Thesepalladium nanoparticles can form growth sites for electrolessmetallization.

It is desirable to alter the optical properties of the microporousmaterial without effecting filtration efficiency. Many metallizationprocesses initially form black deposits (palladium black, black nickel,silver, etc.) This may be due to the very rough (nanoscale)discontinuous nature of the first metallization deposits forming asdiscrete “islands” on the surface, together with surface reactions thatmay involve oxidation, etc. Those skilled in the art of electrolessmetallization are able to choose reaction conditions, reagents, etc. tomaximize formation of black pigmentary deposits within the pores of themicroporous material. These deposits are found to be non-fluorescent andhave minimal effect on membrane flow rate.

In one aspect, the composite includes a microporous material composed ofaluminum oxide and a pigment composed one or more elemental transitionmetals, wherein the pigment is deposited on the microporous material. Inone aspect, the microporous material is Anopore. In another aspect, theelemental transition metal is palladium, nickel, or a combinationthereof with other elements such as phosphorous, boron, or a combinationthereof.

Additionally, it is widely known certain nanoscale metal particlesundergo a process known as Plasmon resonance. In one aspect, thisprocess generally occurs with gold and silver particles in the sizerange of 10 to 200 nanometer dimension. Collections of such particles,when correctly spaced on a surface, may form what may be described as aPlasmon resonance surface. These surfaces can exhibit unique opticalproperties such as enhanced absorption, fluorescence, energy transfer,etc. For instance, it is known Raman scattering of certain molecules canbe enhanced many orders of magnitude in conjunction with Plasmonresonance. Additionally, collections of Plasmon resonance particles,when correctly spaced within a volume, can form what may be described asa Plasmon resonance volume, with unique optical properties. If thePlasmon resonance volume is porous, molecular interactions can occurwithin the large internal Plasmon resonance surface, greatlyfacilitating reaction rates. For example, as disclosed herein nanoscalemetal deposits can be formed within the pores of microporous materials.Under controlled conditions, these deposits can be formed from gold orsilver in a size range and surface density consistent with forming aPlasmon resonance surface. If the microporous material is selected withsuitable pore dimensions and spacing, Plasmon resonance can occurbetween adjacent pores and result in a Plasmon resonance volume.

In one aspect, described herein are methods for producing a pigmentedcomposite. A pigmented composite is any microporous material that has atleast one pigment described herein incorporated in the microporousmaterial. In one aspect, the method involves

(a) contacting a microporous material with a tin compound to produce afirst composite, and(b) contacting the first composite with a pigment comprising anelemental metal, a metal oxide, a metal alloy, a metal salt, or acombination thereof to produce the pigmented composite.Any of the microporous materials and pigments described herein can beused in this aspect.

The tin compound used in this aspect can be any tin compound known inthe art. In one aspect, the tin compound can be an organotin compound ora tin salt. In another aspect, the tin compound can be a compound thatproduces Sn⁺² ions in solution. Examples of tin compounds useful hereininclude, but are not limited to, tin halides such as SnCl₂. Not wishingto be bound by theory, it is believed that the tin compound enhances theadsorption of the pigment onto the microporous material. Additionally,depending upon the pigment selected, the tin compound can react with thepigment. For example, the tin compound can reduce or oxidize a pigment.One advantage of using the tin compound is that it permits the compositeto be rinsed or washed to remove unadsorbed pigment, which ultimatelyresults in a cleaner, more efficient pigmented composite.

The tin compound and pigment can be applied to the microporous materialsusing techniques known in the art including, but not limited dipping,spraying, brushing, etc. In one aspect, independent solutions of the tincompound and pigment are produced, and the microporous material isdipped in the tin solution followed by dipping in the pigment solution.Depending upon the tin compound and pigment that are selected, the tincompound and pigment can be dissolved in water, an organic solvent, or acombination thereof. In one aspect, the tin compound and pigment aredissolved in an organic solvent and optionally diluted with water. Inanother aspect, two or more pigments can be applied to the microporousmaterial after contacting the microporous material with the tincompound. In one aspect, after the microporous material is contactedwith the tin compound, the microporous material is contacted with afirst pigment followed by contacting the first composite with a secondpigment. In an alternate aspect, the microporous material can becontacted with a mixture of two or more pigments. The amount of tincompound and pigment that is used as well as the contacting conditionswill vary depending upon the selection of the tin compound and pigment.

In another aspect, when the microporous material is contacted with asolution of the tin compound and/or pigment, the microporous materialcan be rinsed with water to remove any unadsorbed tin compound andpigment. After the microporous material is contacted with the finalpigment, the pigmented composite can be heated so that the pigment issealed in the microporous material. Not wishing to be bound by theory,it is believed that the heating step shrinks or reduces the size of thepores of the microporous material, which traps or engulfs the pigment.In one aspect, the sealing step can be performed by boiling thepigmented composite in water.

In one aspect, a microporous material such as aluminum oxide iscontacted first with a tin compound such as a tin salt, followed by apalladium compound, and then a nickel compound to produce a pigmentedcomposite. In another aspect, a pigmented composite is composed of amicroporous material, a tin compound, and at least one pigment, whereinthe tin compound and pigment are incorporated in the microporousmaterial.

C. Manipulation of Localized Analytes

Once the analyte is localized on the microporous material, it can befurther processed using techniques known in the art. The followingtechniques are exemplary and are not intended to limit the differenttype of techniques that can be performed.

1. Amplification

In one aspect, when the analyte is a nucleic acid, the localized nucleicacid can be analyzed using amplification techniques including, but notlimited to, polymerase chain reaction, nucleic acid sequence basedamplification, isothermal methodology, ligase chain reaction, and stranddisplacement amplification. Any of the amplification techniquesdisclosed in U.S. Pat. Nos. 4,683,195; 4,683,202; 4,965,188; 5,130,238;5,354,668; 5,427,930; and 5,455,166, which are incorporated by referencein their entireties, can be used in any of the methods described herein.Depending upon the amplification technique used, a specific region ofthe nucleic acid or the entire nucleic acid can be amplified. In oneaspect, when the analyte is irreversibly localized near the surface ofthe microporous material, amplification occurs near the surface of themicroporous material. In another aspect, when the analyte is reversiblylocalized near the surface of the microporous material, amplificationcan occur near the surface of the microporous material and/or insolution where the analyte is no longer localized near the surface ofthe microporous material. In another aspect, localized analytes can beamplified or detected after destabilization of the weak interactionsthat exist between the analyte and the microporous surface by conditionsherein described.

In one aspect, a nucleic acid is amplified by (a) amplifying a nucleicin a fluid sample having a known volume to produce an amplified nucleicacid, and (b) passing the sample comprising the amplified nucleic acidthrough any of the microporous materials described herein, wherein theamplified nucleic acid is localized near the surface of the composite.In another aspect, a nucleic acid is amplified by (a) passing a fluidsample having a known volume comprising the nucleic acid through any ofthe microporous materials described herein, wherein the nucleic acid islocalized near the surface of the composite, and (b) amplifying thenucleic acid.

2. Hybridization and Identification

The following discusses the different type of compounds that can be usedin the hybridization/identification methods disclosed herein.

i. Sequences

There are a variety of sequences that can be used with thehybridization/identification methods disclosed herein, all of which areencoded by nucleic acids or are nucleic acids. The sequences for thehuman analogs of these genes, as well as other analogs, and alleles ofthese genes, and splice variants and other types of variants, areavailable in a variety of protein and gene databases, including Genbank.Those sequences available at the time of filing this application atGenbank are herein incorporated by reference in their entireties as wellas for individual subsequences contained therein. Genbank can beaccessed at http://www.ncbi.nih.gov/entrez/query.fcgi. Those of skill inthe art understand how to resolve sequence discrepancies and differencesand to adjust the compositions and methods relating to a particularsequence to other related sequences. Primers and/or probes can bedesigned for any given sequence given the information disclosed hereinand known in the art.

In certain embodiments this product is at least 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000nucleotides long.

In other embodiments the product is less than or equal to 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58,59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325,350, 375, 400, 425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or4000 nucleotides long.

a. Functional Nucleic Acids

Functional nucleic acids are nucleic acid molecules that have a specificfunction, such as binding a target molecule or catalyzing a specificreaction. Functional nucleic acid molecules can be divided into thefollowing categories, which are not meant to be limiting. For example,functional nucleic acids include antisense molecules, aptamers,ribozymes, triplex forming molecules, and external guide sequences. Thefunctional nucleic acid molecules can act as affectors, inhibitors,modulators, and stimulators of a specific activity possessed by a targetmolecule, or the functional nucleic acid molecules can possess a de novoactivity independent of any other molecules.

Functional nucleic acid molecules can interact with any macromolecule,such as DNA, RNA, polypeptides, or carbohydrate chains. Thus, functionalnucleic acids can interact with the mRNA of any of the disclosed nucleicacids. Often functional nucleic acids are designed to interact withother nucleic acids based on sequence homology between the targetmolecule and the functional nucleic acid molecule. In other situations,the specific recognition between the functional nucleic acid moleculeand the target molecule is not based on sequence homology between thefunctional nucleic acid molecule and the target molecule, but rather isbased on the formation of tertiary structure that allows specificrecognition to take place.

ii. Antibodies

a. Antibodies Generally

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof. Theantibodies can be tested for their desired activity using the in vitroassays described herein, or by analogous methods, after which their invivo therapeutic and/or prophylactic activities are tested according toknown clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces monoclonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro, e.g., using the HIV Env-CD4-co-receptor complexes describedherein.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse.

b. Human Antibodies

The disclosed human antibodies can be prepared using any technique.Examples of techniques for human monoclonal antibody production includethose described by Cole et al. (Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol.,147(1):86-95, 1991). Human antibodies (and fragments thereof) can alsobe produced using phage display libraries (Hoogenboom et al., J. Mol.Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991).

The disclosed human antibodies can also be obtained from transgenicanimals. For example, transgenic, mutant mice that are capable ofproducing a full repertoire of human antibodies, in response toimmunization, have been described (see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ-line antibody gene array intosuch germ-line mutant mice results in the production of human antibodiesupon antigen challenge. Antibodies having the desired activity areselected using Env-CD4-co-receptor complexes as described herein.

c. Humanized Antibodies

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

In one aspect, when the analyte is a nucleic acid, the localized nucleicacid can be hybridized and identified. For example, if a samplecontaining a nucleic acid with an unknown or partly unknown sequence islocalized near the surface of the microporous material or composite andis brought into contact with a binding oligonucleotide that iscomplimentary to the nucleic acid, hybridization will occur. The bindingoligonucleotide can be any naturally-occurring oligonucleotide or amodified oligonucleotide such as, for example, PNA. Based on thehybridization pattern, it is possible to derive sequence informationfrom the localized nucleic acid. Thus, some or all of the nucleic acidsequence can be identified. These hybridization techniques are disclosedin Fodor et al. (1992), Science 251, 767-773 and Southern et al. (1994)Nucleic Acids Res. 22, 1368-1373, which are incorporated by reference intheir entireties.

In one aspect, a nucleic acid is hybridized and/or identified by (a)hybridizing a nucleic acid in a fluid sample with a bindingoligonucleotide having a known sequence that is complimentary to thenucleic acid to produce a hybridized nucleic acid and (b) passing afluid sample comprising the hybridized nucleic acid through any of themicroporous materials described herein, wherein the hybridized nucleicacid is localized near the surface of the composite. In another aspect,a nucleic acid is hybridized by (a) passing a fluid sample comprisingthe nucleic acid through any of the microporous materials describedherein, wherein the nucleic acid is localized near the surface of thecomposite, and (b) hybridizing the nucleic acid with a bindingoligonucleotide that is complimentary to the nucleic acid.

3. Bioreactors

In one aspect, any of the microporous materials described hereinincluding the composites and modified-microporous materials can be usedas a bioreactor. In this aspect, the bioreactor can be used forsmall-size fermentations, biochemical flow analyzers, or a part of abiosensor. In one aspect, a bioactive agent can be covalently attachedto the microporous material either directly or by a linker as describedabove. For example, an enzyme can be attached to the microporousmaterial via silanation described above, which produces anenzyme/support system. In another aspect, the bioactive agent can beattached to the microporous material by way of a suspension matrix toimprove reaction rates. In one aspect, the enzyme/support system canbehave as a catalyst and produce new compounds when a solution ofreactants are contacted with the enzyme/support system. In anotheraspect, the enzyme/support system can behave as a biosensor and interactwith specific analytes present in a sample.

4. Quantification

In one aspect, the localized analyte can be used to quantify the amountof analyte that was in the fluid sample. The phrase “quantifying ananalyte” is defined herein as calculating the amount of analyte presentin a known volume of sample once the analyte has been localized near thesurface of the microporous material. In one aspect, the analyte isquantified by detecting and counting the analyte particles andcorrelating the number of analyte particles to a correspondingconcentration based on the known volume of the sample.

The methods described herein for quantifying analytes permit the rapidanalysis of the analyte. In one aspect, the analysis of the analyte canbe performed in less than 30 minutes. In another aspect, the analysis ofthe analytes do not require target molecular amplification ormultiplication (e.g., polymerase chain reaction (PCR)) for highsensitivity, which provides improved precision and accuracy. Numerousother advantages are described below.

i. Labeling of Analyte

Depending upon the detection technique used, in one aspect, the analytecan be labeled prior to detection. The term “labeled analyte” as usedherein is defined as an analyte that has interacted with a detectabletracer. The interaction between the analyte and the detectable tracercan include any chemical or physical interaction including, but notlimited to, a covalent bond, an ionic interaction, or a Lewis acid-Lewisbase interaction. A “detectable tracer” as referred to herein is definedas any compound that (1) has at least one group that can interact withthe analyte as described above and (2) has at least one group that iscapable of detection using techniques known in the art. In one aspect,the analytes can be labeled prior to localization. In another aspect,the analyte can be labeled after it has been localized.

When the analyte is a nucleic acid, techniques for modifying nucleicacids to allow binding and labeling through specific hybridization arewell known in the art. For example, target nucleic acids may belocalized to a surface specifically or nonspecifically. The phrase“specifically localized target nucleic acid” is defined herein as targetnucleic acids present in a sample that are specifically localized nearthe surface of the microporous material, for instance by specificbinding reactions that only capture and localize the specific targetnucleic acid. Any target nucleic acids not specifically localized arereferred to herein as “non-specifically localized target nucleic acids.”Labeling for pure, specifically localized target nucleic acids, whereonly target nucleic acids are localized, may be accomplished by usingcommercially available molecular labels, such as fluorescent nucleicacid dyes. In one aspect, the localization of specifically localizedtarget nucleic acids can be accomplished by employing a localizedspecific binder, such as a complimentary binding oligonucleotide, tospecifically capture and localize a target nucleic acid to near thesurface of the microporous material. In this aspect, all nucleic acidslocalized near the surface of the microporous material are targetmolecules and can be labeled and counted.

In another aspect, non-specifically localized target nucleic acidlocalization involves localizing most nucleic acids contained within thepatient sample near the surface of the microporous material. In thisaspect, labeling non-specifically localized target nucleic acids, wheretarget and other nucleic acids are co-localized to the surface, may beaccomplished either by using a detectable tracer such as a specificbinding probe labeled with detectable markers, or by forming surfacelocalized hybrids between the localized target nucleic acids andspecific binding probes with subsequent labeling such as withfluorescent nucleic acid dyes. In this aspect, target molecules can bespecifically labeled and counted in the presence of potentially highconcentrations of unwanted nucleic acids.

In one aspect, localized virion-derived nucleic acids can befluorescently stained with non-specific nucleic acid dyes.Alternatively, the nucleic acid dye may be added to the lysed patientsample filtrate prior to the localization step. The localizedfluorescent nucleic acids can then be counted using the techniquesdescribed herein. This approach provides an improved technique toidentify potentially infectious particles.

In one aspect, the detectable tracer is two or more different detectabletracer molecules. Examples of detectable groups present on thedetectable tracer include, but are not limited to, a fluorescentmicrobead, a quantum dot, a surface plasmon resonance particle, afluorescence generating enzyme, a fluorescent dye, or a combinationthereof. In one aspect, the detectable tracer is a labeled ornon-labeled oligonucleotide probe. In another aspect, the followingdetectable tracers can be used to label nucleic acids.

a. Fluorescent Tracers

Fluorescent nucleic acid staining can be used for labeling targetnucleic acid molecules. Fluorescent nucleic acid dyes selectively stainnucleic acids through intercalation, minor groove binding, etc. The mostuseful of these dyes exhibit a strong fluorescence enhancement onbinding to nucleic acids. Non-limiting examples of such dyes includeethidium bromide, propidium iodide, Sybergreen I, Toto-3, Sytox Orange,and the like. Some of these dyes have high quantum yields (greater thanabout 50%) and fluorescence enhancements of greater than 1000 uponbinding to certain types of nucleic acids. This fluorescence enhancementupon binding to nucleic acid minimizes the signal-to-noise limitationsof fluorescent tracers, and dye nonspecific binding is essentiallynonfluorescent. Additionally, dye loading per target nucleic acid can behigh, often approaching 1 fluorescent dye molecule per 4 base pairs.Limitations of fluorescent nucleic acid dyes include staining of allnucleic acids on the target surface, although frequently with apreference for double strand DNA, RNA, etc.

Additionally, a sequential fluorescent staining approach may be used.Such an approach is especially useful in cases where although targetnucleic acids are specifically localized near the surface of themicroporous material with complimentary oligonucleotides, it is unlikelythat all nucleic acids on the surface will be target nucleic acids tothe level of purity required for biological assay. In this case, targetand non-target nucleic acids are co-localized. In the sequentialstaining approach, a first nucleic acid dye A is added thatpreferentially stains the double stranded segments of all localizednucleic acids with very high affinity. Free dye A is then removed fromthe system, and a complimentary oligonucleotide probe (unlabeled) isadded and binds to the target nucleic acid. A second nucleic acid dye Bis then added and binds to the newly created oligo probe-target nucleicacid hybrid regions. This results in the true target nucleic acid beinglabeled with dye B while dye A blocks all nonspecific nucleic acid dyebinding sites.

DNA, RNA, and PNA (peptide nucleic acid) oligonucleotide based probescan be used to label target nucleic acids either before or afterlocalization through specific hybridization. The phrase “surfacelocalized hybrid” is defined herein as the product formed when a nucleicacid localized near the surface of the microporous material is broughtinto contact with a substrate (e.g., DNA or RNA) having a known sequenceso that the substrate interacts with the localized nucleic acid.Alternatively, the phrase “surface localized hybrid” is defined hereinas the product formed when a nucleic acid is brought into contact with asubstrate (e.g., DNA or RNA) having a known sequence so that thesubstrate interacts with the nucleic acid to produce a hybrid and thenlocalizing the hybrid. Many such systems are known to those skilled inthe art. These hybridization probes can be used to form detectabletracers by direct labeling with materials to make them opticallydetectable. Examples of probes include, but are not limited to, dyes,beads, proteins and protein aggregates, quantum dots, nanocrystals, andthe like. Additionally, the nucleic acid can be labeled with materialsto make them optically detectable after additional steps. For instance,biotinylated probes are widely used in molecular diagnostics. Afterhybridization, the bound biotinylated probes are reacted with opticallydetectable materials containing specific binders for biotin, forinstance avidin, streptavidin, neutravidin, etc. The hybridizationprobes may additionally be directly or indirectly labeled with enzymesthat are able to create optical signals after additional steps as willbe described later.

In one aspect, viral load assays can be labeled. HIV contains singlestrand ribonucleic acid (RNA) approximately 9,000 bases in length. Thereare 360 separate 25 base length oligonucleotide probes that can begenerated against the complete HIV RNA molecule. The ability tosynthesize numerous, different oligonucleotide probes for specificbinding to HIV RNA, as well as other target nucleic acids, is well knownby those skilled in the art. Likewise, the ability to make thesespecific oligo probes fluorescent through incorporation or attachment ofdyes, fluorescent beads, fluorescence generating enzymes, etc. is alsowell known.

The choice of fluorescent label for attachment to oligonucleotide probesto produce a detectable tracer is largely determined by detectorparameters. Example 14 sets forth present detection data calculationsbased on detecting 25 or 100 highly fluorescent molecules (e.g.,fluorescein, rhodamine, Cy 5, etc) per target nucleic acid with a lowpower scanning detection system such as a scanning confocalepifluorometer. In one aspect, a process for producing such labeledmolecules is as follows. Localized target nucleic acids are incubatedwith probe solution to allow hybridization to occur. In the case ofporous membrane localization, flowing probe solution through themembrane during hybridization can shorten reaction times. Unhybridizedprobe solution is rinsed from the optical detection surface underconditions to control hybridization stringency, NSB, etc.

A fluorescent bead based tracer process is depicted schematically inFIG. 3. Initially, fluorescent particles such as fluorescent microbeads300 are coated with specific binding oligonucleotide probes which arelocalized on the microbeads. The fluorescent microbeads 300 are sizedfor retention on the surface of a membrane filter 310. After the targetnucleic acids have been localized near the surface of membrane filter310, the fluorescent microbeads are introduced and filtered onto thesurface of membrane filter 310. The fluorescent microbeads, coated withbinding oligonucleotides, are thus placed in direct contact with thelocalized target nucleic acids, and hybridization reactions rapidlyoccur unimpeded by diffusion. After a suitable incubation period,unbound microbeads 320 are rinsed off under conditions that controlhybridization stringency and binding specificity. Multiple beads shouldbe attached to each target nucleic acid to discriminate against tracerbead nonspecific binding.

b. Enzyme Tracers

In another embodiment, enzymatic techniques can be used as a detectabletracer to label analytes with high SNR. The principle of enhancedactivity of “suspended” reactants can be used. For example, antibodiesand other proteins, as well as other molecules, are known to loseactivity when retained to a surface. Retention of analytes to a“scaffolding” matrix, for instance of macromolecules localized to amicroporous material can greatly improve activity by minimizing surfaceinactivation and improving mass transfer by allowing flow through theactive microporous material.

Fluorescent precipitation assays have been described in the art and usean enzyme tracer that converts a soluble, non-fluorescent substrate to afluorescent precipitate localized at the point of enzymatic activity.Examples of enzyme tracers used in precipitation assays include, but arenot limited to, alkaline phosphatase as used in Enzyme LabeledFluorescence (ELF), which is available from Molecular Probes, Inc. Inthis case, localized target nucleic acids are hybridized tooligonucleotide probes labeled with suitable enzymes that subsequentlycreate localized fluorescent microprecipitates that can be counted inone-to-one correspondence to the target nucleic acids.

FIG. 4 shows an ELF reaction used herein. Target nucleic acid 400 islocalized near the surface of microporous material 401 and physicallyspans several openings 402. Biotinylated hybridization probe 403 hasbeen specifically hybridized to target nucleic acid 400. Note the figureis not to scale, as the typical 25 base nucleotide probe is only about 8nanometers long, compared to the nominal pore diameter of approximately180 nanometers. Alkaline phosphatase-streptavidin conjugate 404 has beenbound to biotinylated probe 403. Unbound probes and unbound conjugateshave been rinsed from the system. Nonfluorescent, soluble ELF substrate405 has been added and conjugate 404 is generating fluorescentprecipitate 406 on and within filter 401. In this case, conjugate 404bound to hybridized probe 403 is typically “suspended” over a pore ofthe surface. This typically results in high enzymatic activity due tominimal surface denaturation and excellent multidirectional substratediffusion. Nonspecifically bound conjugate 407 present on the membranesurface is typically much less active due to surface denaturation of theconjugate as well as relatively inhibited substrate diffusion to thesurface. Additionally, the ELF fluorescent precipitate 406 is generatednear the surface of the microporous material. This enhances nanocrystalprecipitation, localizes the nanocrystal near the surface, and somewhatlimits nanocrystal growth to typical pore dimensions. All thesecontribute to improved detectability. As previously discussed, multipletracer binding may additionally be used to improve SNR.

FIG. 5 shows a graph of the timing of a typical ELF reaction using themethods described herein. The horizontal axis is time and the verticalaxis is the local concentration of the ELF precipitatingdephosphorylated substrate, also known as the ELF alcohol. A dashedhorizontal line, labeled concentration at precipitate formation 510represents the effective concentration where nanocrystal precipitationwill begin. Two diagonal lines are present. The steepest slope line islabeled Specific Signal 511, while the shallow slope line is labeled NonSpecific Signal 512. These two lines represent the local ELF alcoholconcentration present near locations of high (steep slope) and low(shallow slope) enzymatic activity versus time. Locations of lowenzymatic activity may be non-specifically bound conjugates aspreviously described, while high enzymatic activity locations may bespecifically bound enzymes, either due to uninhibited single moleculeenzyme activity or multiple probe and enzyme binding in a small area (1micron²). In this system, fluorescent nanocrystals cannot form until thelocal concentration of the ELF alcohol exceeds the concentrationindicated by line 510. As is seen, at some time into the reactiondesignated Initial Precipitation Time 513, only the locations ofrelatively high enzymatic activity have generated sufficient ELF alcoholto cause nanocrystal formation. At later times, even regions of lowenzymatic activity can eventually produce nanocrystals. Accordingly, ahigh SNR for specifically versus non specifically bound probes existsrelatively early in the course of the reaction.

FIG. 6 is a fluorescent spot count versus time graph for nucleic acidcounting as described in Example 22. Briefly, lambda phage DNA wasprehybridized with 20 complementary biotinylated probes, localized to animproved Nickel-Boron Anopore membrane as described herein, anddeveloped with ELF as described in the Example. As is seen, a cleardifferential of lambda positive versus lambda negative samples wasdetected, especially very early in the course of the reaction.

Similarly, tyramide based assays enzymatically generate reactivefluorescent intermediates that covalently attach to proteins near thepoint of enzymatic activity and may be configured to produce localizedmicrofluorescent zones that may be counted in one-to-one correspondenceto the target nucleic acids.

c. Modified Microporous Materials for Enhanced Reaction Rates

In another aspect, the enhanced reaction rates of suspended reactantscan be beneficially employed. In one aspect, a suspension matrix islocalized near the surface of the microporous material, including anycomposite described herein, to produce a modified microporous material.In one aspect, the modified-microporous material can be produced bycontacting the microporous material with a solution containing thesuspension matrix, wherein the solution passes through the microporousmaterial and the suspension matrix remains localized near the surface ofthe microporous material. In another aspect, the suspension matrix isadded to a sample containing the analyte prior to contacting the samplewith the microporous material. In this embodiment, the suspension matrixinteracts with the analyte prior to localization near the surface of themicroporous material. The suspension matrix can interact with theanalyte via a covalent bond or any physical or ionic interaction.

One aspect of this is depicted in FIGS. 7 a-7 c. Microporous material700 containing micropores 710 is shown in FIG. 7 a. FIG. 7 b showssuspension matrix 720 localized near the surface of the microporoussurface 700 spanning over micropores 710. Suspension matrix 720 isattached to the microporous material 700 sufficiently to preventmolecular collapse through micropores 710 as disclosed herein. In oneaspect, the suspension matrix can be localized to the microporousmaterial by, for example, covalent attachment, entrapment, or physicalor ionic interactions. FIG. 7 c shows a cross section throughmicroporous surface 700 showing suspension matrix 720 “suspended” overmicropores 710. Suspended analytes 730 are shown attached to suspensionmatrix 720 over micropores 710, and non-suspended analytes 740 attachedto suspension matrix 720, not over micropores 710 but over the solidsurface support of microporous material generally shown as 750. Asdisclosed, suspended analytes 730 have enhanced reaction rates based onsteric hindrance, diffusion, denaturation, etc. compared to nonsuspended analytes either on the suspension matrix but not suspendedover a micropore, as shown at 740, or non-specifically bound orconventionally localized analytes (directly to the surface), as shown at760.

The suspension matrix can be any macromolecule or similar structurecapable of spanning the micropores on the microporous material. Examplesinclude, but are not limited to, organic or inorganic macromolecules,polymers, nanofibers, etc. of sufficient size so as to efficientlylocalize to the microporous surface. In one aspect, the suspensionmatrix can be a polymer or macromolecule, such as an oligonucleotide, apolysaccharide, or a protein. In one aspect, the suspension polymer is anucleic acid such as DNA and analogs thereof and RNA. In another aspect,the suspension polymer is a polysaccharide such as cellulose and starch.Reactants can be proteins or other molecules including, but not limitedto, enzymes and antibodies, biomolecules, or catalysts. The analytes canbe directly attached to the suspension matrix, for instance throughcovalent bonds, through a linker, or a binding reaction such asbiotin-avidin. The analytes can be attached to the suspension matrixbefore or after it is attached to the microporous material. For example,antibodies can be attached to DNA in solution by methods generally knownin the art, then the DNA, containing the attached antibodies, islocalized to a microporous surface. Part of the attached antibodies willbe suspended over the micropores and have enhanced reactivity. If theDNA is reversibly localized to the surface, it may be removed by methodsdisclosed herein, thereby additionally removing the attached antibodies.In another aspect, the DNA containing the antibodies can be reacted insolution with an analyte or tracer of interest to affect specificbinding. Here, the DNA-containing attached antibodies and a specificallybound analyte or tracer can then be localized to the microporous surfacefor enhanced tracer detection as disclosed herein. In this case, theease of concentrating reactants, for instance antibodies, analytes, andtracers, from solution by localization of a suspension matrix to amicroporous material regardless of the relative activity enhancement ofsuspended analytes is another advantage of this method.

d. Plasmon Resonant Assemblies

Plasmon Resonant Particles (PRP) are metallic submicron particles thatdisplay unusual optical properties. In particular, gold and silver PRPsare known to have exceptionally high and wavelength selective lightscattering ability. Indeed, a single 60 nanometer gold particle scattersthe equivalent photons as the fluorescence emission of 500,000fluorescein molecules. Silver PRPs are known to be approximately 8 timesas efficient as gold although chemically less stable. PRPs have beenproposed for use in diagnostics and several companies have been formedto commercialize PRP and related technology.

Spherical PRP optical properties are well known:

1. For very small PRPs (under 40 nm for gold), the scattering crosssection is proportional to the particle radius to the sixth power, whilethe peak scattering wavelength remains constant. In this regime, goldscatters primarily at 520 nm, while silver scatters at 380 nm.

2. As the PRPs become larger (greater than 40 nm for gold), thescattering cross section continues to increase while the peak scatteringwavelength shifts to longer wavelengths. For example, the followingscattered colors are apparent:

Gold PRP Diameter Scattered Color  40 nm green  78 nm yellow 118 nmorange 140 nm red

3. Unlike transparent dielectric scattering particles (glass, plasticbeads, etc.), PRPs show no reduction of scattering parameters with mediarefractive index matching. Indeed, while scattering from conventionalparticles may be essentially eliminated by matching the immersion mediarefractive index to the intrinsic particle refractive index, scatteringfrom PRPs increase with increasing media refractive index as well asundergo a distinct red shift in peak scattering wavelength.

Characterization of non-spherical (rod shaped, etc.) PRPs areconsiderably less developed. Published data on rod shaped PRPs indicateessentially a two axis scattering parameter, where scattering wavelengthdepends on the instantaneous PRP orientation to the illumination light.For example, PRP rods with a diameter of less than 40 nm display a 520nm scattering peak and a potentially stronger, longer wavelength peakassociated with their length.

Characterization of assemblies of spherical PRPs indicate resonantinteraction between closely spaced PRPs. That is to say, a linear arrayassembly of closely spaced spherical PRPs shows long wavelengthscattering characteristics similar to an equivalent rod. An assembly oftwo closely spaced particles will show a fundamental scatteringwavelength based on PRP diameter (for instance, 520 nm for <40 nm gold)as well as a longer wavelength associated with resonant interactionbetween the mutually resonant Plasmon states. A linear assembly of threePRPs will show the fundamental wavelength as well as a unique longerwavelength associated with the long dimension of the three spherical PRPassembly. Much like FRET, this resonant assembly of PRPs is a veryshort-range process and may be useful for exacting analysis of proximitybinding events.

In the methods described herein, when the analyte is a nucleic acid,plasmon resonant assembly (PRA) detection may allow nucleic acidstructural analysis. As previously described, a detectable tracer suchas multiple probe binding can be used to distinguish specific fromnon-specific binding events. While the above described multiple probetechnique successfully allows discrimination of (NSB) from true targetmolecules, nucleic acid sequence information is somewhat less certain.Assume a target molecule with a single nucleotide polymorphism (SNP).Additionally, assume 10 probes are synthesized to specifically bind tothe 250 base region containing the SNP (assume 10 probes each 25 baseslong). Each probe contains a fluorescent reporter means (microbead,quantum dot, highly fluorescent tag, biotin for sequential labeling,etc.) The probe actually coding for the SNP displays measurable andpredictable binding deviation (melt temperature, etc.) when compared tothe binding on a non-SNP target molecule. Theoretically, it is possibleto identify the SNP by anomalous fluorescence intensity changes duringprobe-target molecule melting or binding under high stringencyconditions. Practically speaking, Nucleic Acid Counting (NAC) detectorsoperate with comparatively low signal to noise ratios consistent withdigital molecular detection and cannot reliably detect a 1 of 10deviation in fluorescence intensity as described in the above example.Fewer probes (say 3 rather than 10) certainly make the fluorescentintensity differences greater for SNP detection, but potentiallyincrease false positives due to NSB.

It would be advantageous if multiple probe binding could be specificallyand unambiguously detected at low signal to noise ratios. For instance,fluorescent resonant energy transfer (FRET) is widely used, whereinmultiple binding within a small region is detected by unique wavelengthemission. A similar system, but with characteristics amenable to NACdetection (high detectability, unique multiprobe signature, etc) mayallow SNP identification without molecular multiplication.

PRA detection is capable of distinguishing assemblies of multiple (2, 3,4 etc.) PRPs uniquely based on optical scattering parameters. Forexample, a 5-probe system can be used for SNP detection. In one aspect,the probes can be designed to be contiguous with the SNP location underthe center (#3) probe. In this case, an assembly of 5 PRPs undergoingpredictable melting in the absence of the SNP and an assembly of 5 PRPsmelting into 2 assemblies of 2 PRPs for nucleic acids containing the SNPin question is expected. The loss of #3 probe during melting results ina major scattering wavelength shift from the far red (1×5-40 nm gold PRPassembly) to yellow (2×2-40 nm gold PRP assembly). Other schemes arepossible, depending on spherical PRP diameter, material, and PRPassembly parameters. Additionally, it is well known silver enhancementcan be used to “grow” PRPs in solution starting with very small goldparticles. In one aspect, these small gold particles (<2 nm diameter)may prove less prone to NSB on Anopore membranes. Silver enhancement ofPRAs, based on small gold nucleation sites may result in more rod likestructures and unique detection.

ii. Detection

Once the target analyte is localized, the analyte can then be detectedby a detection system. In one aspect, the localized analyte is labeled.Depending upon the detection technique, the analyte can be labeled priorto or after localization. In one aspect, as known in the art, an analogtype detection scheme may be employed, where labeled analytes eachcontribute to an ensemble signal that needs to be of sufficient strengthfor detection. In another aspect, a digital molecular counting (MC) ordigital nucleic acid counting (NAC) detection system can be used tointerrogate molecule size zones on the assay target surface for thepresence or absence of fluorescence.

Digital detection of the localized analyte can be accomplished with highsensitivity detectors, such as high sensitivity, high spatial resolutionfluorescent detectors that interrogate the surface of the microporousmaterial in small regions (e.g, about 1-10 micron areas) for thepresence or absence of fluorescent signal from the labeled analytes. Theinterrogation area is sufficiently small, and the localized analytesurface concentration is sufficiently low so as to practically allowonly 0 or 1 labeled analytes within the interrogation area. An analyteis considered counted when the detected signal exceeds a preset ordynamically calculated threshold determined by the relative magnitude ofthe fluorescent signal from the labeled target analyte and thebackground signal.

In another aspect, detection of the analyte can be accomplished withother techniques including, but not limited to, fluorescence,phosphorescence, chemilumenescence, bioluminescence, Raman spectroscopy,optical scatter analysis, plasmon resonant particle (PRP) analysis, etc.and other techniques generally known to those skilled in the art.

In the case of digital nucleic acid detection, some or all of thefollowing nucleic acid detection requirements should be met depending onthe type of detection system utilized.

1. Unambiguously isolate the fluorescence from an interrogation zoneapproximately 1 to 10 microns in area.

2. Measure fluorescent emissions from the interrogation zone with highefficiency and sensitivity consistent with the fluorescent labelingsystem used.

3. Measure approximately 5,000 to 500,000 discrete interrogation zonesper second.

4. Maintain focus on the target surface during measurement.

5. Maintain intensity-location relationship (image formation) forbackground subtraction.

Examples of suitable detection devices that can be used herein include,but are not limited to, high speed scanning confocal epifluorometers,high sensitivity fluorescent confocal microscopes, charge coupled device(CCD) arrays such as CCD array based fluorescent microscopes, imageintensified cameras, and the like. High speed scanning confocalepifluorometers are able to detect a few fluorescent labels on eachnucleic acid. Several labeling schemes previously discussed (e.g.,fluorescent microbeads, nucleic acid dyes, enzymes, quantum dots, etc.)produce substantially greater signal and are detectable with lesssophisticated equipment.

Recent advances in ultra high sensitivity optical detection haveresulted in the ability to identify single fluorescent molecules, whichpermits numerical counting of single molecules localized to surfaces. Inone aspect, a labeled analyte that is localized near the surface of themicroporous material can be detected by optical detection. Opticaldetection generally involves detection methods employing electromagneticradiation in the wavelength range of about 200 nm to about 20,000 nm. Inone aspect, for ultra low concentration nucleic acid detection, such asfound in ultra sensitive viral load assays, and the like, opticaldetection can be used in any of the methods described herein for thedirect determination of nucleic acid concentrations without molecularamplification.

In one aspect, when a sequential fluorescent staining approach asdescribed above is used to label a localized nucleic acid, and the twodyes (A and B) are the same, the optical surface is measured twice, onceafter initial staining and again after complimentary specific bindingoligonucleotide probe addition. In one aspect, the detector used in thiscase is an imaging detector such as a confocal fluorescent microscope,CCD camera, or the like. The images obtained from these respectivemeasurements are simply subtracted to derive the true target nucleicacid count. This is true even if the true target nucleic acid partiallystains during the initial staining.

An example of this type of technique is an HIV viral load nucleic aciddetection assay using the dye Picogreen. This dye is known topreferentially stain double strand DNA or DNA-RNA hybrids. The nucleicacids in a patient sample, including single strand HIV RNA, arelocalized near the surface of a microporous material, such as an Anoporemembrane. Picogreen solution is added and fluorescently stains alllocalized double strand nucleic acids, including self hybridized(hairpin, etc) regions of the HIV RNA. The membrane filter isfluorescently imaged to produce a background image measurement.Oligonucleotide probes, complimentary to the HIV RNA, are added andallowed to specifically hybridize to the single strand HIV RNA.Picogreen solution is again added (or simply not removed from the firststaining and hybridization steps) and the membrane filter is reimaged.The previously taken background image is subtracted from this secondimage to create a difference image. This difference image represents theHIV RNA regions that specifically hybridized with the complimentaryoligonucleotide probes. Target HIV RNA is then detected as fluorescentspots or pixels on the difference image.

In the example above, if dyes A and B are spectrally independent, onlythe dye B measurement is required. Dye A may be nonfluorescent so thatit simply blocks non-target nucleic acid binding sites for fluorescentdye B. In this case, non-imaging detectors may be employed for nucleicacid detection since fluorescent images need not be subtracted.

a. Scanning Confocal Epifluorometer

One aspect of a suitable scanning detection system for use in molecularcounting is shown schematically in FIG. 8. The detection system,designated generally as 800, includes a laser light source 802 that isin optical communication with a light beam delivery and collectionmodule 804, which can include various optical components for directinglaser light to the sample to be measured and collecting the fluorescedlight. As shown in FIG. 8, the optical components of delivery andcollection module 804 include a collimating lens 806, a fluorescencebeam splitter 808, a polarizing beam splitter 810, a quarter wave plate812, a dynamic focus objective lens 813, and a focus detector 814. Thefocus detector 814 includes a focus lens 815, a cylindrical lens 816,and a quadrant photodiode detector 817. The interaction of these opticalcomponents with a laser beam from light source 802 will be discussed infurther detail below. The delivery and collection module 804 is inoptical communication with a detector module 818, which includes aninterference filter 820, a first focus lens 822, a spatial filter 824, asecond focus lens 826, and a photon counting avalanche photodiode (APD)828.

During operation of detection system 800, a laser beam 830, such as alinearly polarized continuous wave (cw) laser beam, is expanded andcollimated to a preselected diameter, such as up to about a 6 mmdiameter. The laser beam traverses fluorescence beam splitter 808,polarizing beam splitter 810, quarter wave plate 812, and is focusedonto the interior surface of sample container 832 by dynamic focusobjective lens 813. The fluorescence beam splitter 808 transmits laserbeam 830 and reflects all other wavelengths of light. The fluorescencebeam splitter 808 can be set at an angle such as approximately 15degrees incidence to minimize polarization effects and improve thesignal-to-noise ratio. The polarizing beam splitter 810, in conjunctionwith quarter wave plate 812, forms a traditional optical isolator. Thelinearly polarized laser light traverses polarizing beam splitter 810and is converted into circularly polarized light by quarter wave plate812. Specular reflection of this light reverses the direction of thecircular polarization (right hand into left hand circularization). Thisopposite hand polarized light is converted into linearly polarized lightupon returning through quarter wave plate 812. This return light islinearly polarized orthogonally to the original laser beam, and isreflected at 90 degrees through polarizing beam splitter 810 to focusdetector 814.

The dynamic focus objective lens 813 maintains laser beam andfluorescence detector focus on the target surface during scanning.Scanning can be accomplished by rotating sample container 832 (e.g.,disposable polystyrene culture tubes, inorganic optical membranefilters, etc.) while moving the container or detector along thecontainer axis. Alternative scanning schemes use scanning mirrors (notshown) between quarter wave plate 812 and objective lens 813 to scan inorthogonal directions. The objective lens numerical aperture (NA) can belimited to about 0.6 to allow enough depth of focus for the auto focussystem to work, as well as allow a convenient working distance betweenobjective lens 813 and sample container 832.

The objective lens 813 can be placed in a linear motor (voice coil) andtranslated towards or away from sample container 832 under closed loopcontrol of the focus system to maintain focus on the interior surface ofsample container 832 during scanning. As is well known to those skilledin optical design, optical reflection will occur at the interfacebetween two transparent materials of different refractive indexes. Forexample, the reflection from a glass-to-water interface is approximately0.5%, while the reflection from a glass-to-air interface isapproximately 4%. The detection system 800 can be configured todynamically focus on a dry surface (interface), or on the interfaceformed between a solid and a fluid. Accordingly, the weakly reflectedlaser light (about 0.5%) from the container-fluid interface traversescylindrical lens 816 of focus detector 814 and illuminates photodiodedetector 817. The optics are such that when objective lens 813 is infocus with the container-fluid interface, a circular spot is generatedon focus detector 814. Deviation from the ideal focus location resultsin elongation of the circular spot. This elongation is measured byphotodiode detector 817 and used as an error signal in a feedback loopfocus controller for objective lens 813.

Fluorescence from the interior surface of sample container 832 follows areturn path along that of the laser excitation and is reflected byfluorescence beam splitter 808. In this location, most specularlyreflected excitation light has been removed from the fluorescent signalby polarizing beam splitter 810. Depending on fluorescence polarization,some fluorescent signal may also be lost. In an alternativeconfiguration, fluorescence beam splitter 808 may be located betweenquarter wave plate 812 and objective lens 813. In this alternativelocation, the fluorescent signal will be greatest, but also will haveadditional reflected excitation light. As shown in FIG. 8, a reflectedfluorescence beam 840 from fluorescence beam splitter 808 traversesinference filter 820, which can be a very high efficiency, multicavityinterference filter. The fluorescence beam 840 is focused onto spatialfilter 824 by lens 822. The spatial filter 824 is located at an imageplane of the container-fluid surface and improves spatial resolution andsignal-to-noise ratio by eliminating light from other areas. The lighttraversing spatial filter 824 is focused by lens 826 onto a small areaof photon counting avalanche photodiode 828. Depending on the activephotodiode area and image magnification, the spatial filter system maybe simplified by directly focusing the fluorescent light onto a smallarea photodiode. As is well known to those skilled in the art, imagesmay be constructed by tracking signal intensity versus scan position.Such images can be visually displayed on an output display such as acomputer monitor or the like, or further analyzed by a computer to countthe detected nucleic acids. It will be understood by those skilled inthe art that detection system 800 may be configured for use with manydifferent geometries, such as filter membranes, flowcells, multiwellplates, and the like.

b. Camera Based Dark Field Fluorometer

Another suitable detection system that can be used in the molecularcounting methods described herein is shown schematically in FIG. 9. Thedetection system, designated generally as 900, is particularly suitedfor detecting nucleic acids localized on a microporous material. Thedetection system 900 includes a light source 902 including excitationfilter 950, that is in optical communication with a detector module 904such as a charge coupled device (CCD) camera. The detector module 904includes one or more interference filters 906 to isolate labeled nucleicacid fluorescence. The detector module 904 can have a flat fieldobjective lens 908, such as a lens having an NA of about 0.1 to about0.3 to achieve a depth of focus of about 5 microns to about 15 microns.This large depth of focus allows latitude in sample filter membranelocation. Alternatively, higher NA objective lenses may be used tocollect additional signal, but with smaller depth of focus. The detectormodule also includes a detector lens 910, and CCD array 912, such as acooled high quantum efficiency back thinned CCD detector, for exposuresof several seconds to several minutes. Other optical designs involvingthrough the lens and fiber optic illumination can also be employed inalternative embodiments.

During operation of detection system 900, an illumination beam 920 isdirected to the surface of the microporous material 930 having nucleicacids localized thereon. The illumination beam 900 causes fluorescentlylabeled analyte to fluoresce. A localized analyte, even labeled withmany fluorescent microbeads, is still a very small object. Each labeledanalyte will typically generate a single bright pixel on CCD array 912from a fluorescence beam 940. Assuming detector system 900 is designedfor a 3 mm diameter target surface with a 1000×1000 CCD array, eachpixel represents an area of about 3×3 microns on the membrane filter.The detector system 900, unlike the previously described scanningconfocal system of FIG. 8, can be designed for analytically uniformillumination and detection. This should allow pixel signal intensity tobe correlated with fluorescent nucleic acid concentration per pixel.Accordingly, closely spaced analytes (within the same pixel) may beresolvable by step functions in pixel intensity, improving detectorcounting dynamic range. The images produced by the CCD array can bevisually displayed on an output display such as a computer monitor, orfurther analyzed by a computer to count the detected analytes.

c. Scanning Darkfield Spectrometer

In one aspect, a detector suitable for molecular counting based onPlasmon resonance assembly (MC-PRA) detection is shown in FIG. 10 andgenerally identified as 1000. It is best characterized as a dark field,confocal scanning spectrometer. Unlike fluorescent-based systemspreviously described for molecular counting, this detector operates withsubstantial light levels and does not require high sensitivitydetection.

Illumination should be from a white light source and be positioned suchthat direct reflections are not detected by the detector. This istypically accomplished using a high intensity, compact arc lamp withsuitable optics to illuminate the microporous material at an anglegreater than the total collection angle of the detection objective. Thismay be done with conventional microscope dark field illuminationequipment or dark field metallographic microscope objectives and relatedhardware. Compact arc lamp 1001 emits broadband (multiwavelength) lightwhich is collimated by condenser lens 1002. This light impinges onobscuration disc 1003, which forms annular illumination beam 1004. Thisannular beam of illumination light is reflected by mirror 1005,containing a central hole for detected light to pass through. Theannular illumination is reflected by annular reflector 1006 at an anglegreater than the maximum optical collection angle of objective 1007 toilluminate nucleic acids containing suitable labels 1009 localized onthe microporous material 1010. In this way, specularly reflectedillumination light, for instance from a first surface reflection fromthe microporous material, cannot enter the collection optics. Opticalsignal is collected by objective 1007 relatively free from first surfacereflection and passes through the hole in mirror 1005 and is imaged ontoconfocal pinhole 1013 by lens 1012. Confocal pinhole 1013 assists inlowering spurious scattered light, etc. The light that traverses thepinhole is directed to concave grating 1014, where it is dispersed intoseparate wavelengths 1015 and is detected by photodiode array 1016. Thisarray simultaneously detects multiple wavelengths present in thescattered signal from the PRA labeled nucleic acids. As previouslydiscussed, the intensity of these scattered wavelengths may be used todetermine labeling parameters of the localized nucleic acids. In thisexample, scanning may be accomplished by moving the membrane assemblyunder a stationary detector as shown by 1017, or alternatively movingthe detector assembly while keeping the membrane assembly stationary.

Scattered light from the PRA originates within a very small area nearthe surface of the microporous material. It is advantageous to employhigh numerical objectives and confocal geometry to limit extraneousscatter from reaching the detector.

The MC-PRA detector should simultaneously detect multiple wavelengths.FIG. 10 depicts one embodiment of this, where it is accomplished byemploying an optical grating to disperse the scattered light traversingthe confocal pinhole onto a linear array photo detector. Light levelsare sufficiently high to allow conventional photodiode detectors ratherthan more expensive photon counting and cooled CCD systems.

d. Melting Curve Analysis

When the analyte is a nucleic acid, melting curve analysis (MCA) is atechnique based on the reversible nature of nucleic acid hybridization.Double strand nucleic acids, as well as single strand nucleic acids withextensive secondary structure (self hybridization), revert to simplesingle strand configurations under well-defined conditions oftemperature and solvent composition. This process is referred to asmelting. Many nucleic acid stains show large fluorescence enhancementwhen bound to double strand nucleic acid segments, and will losefluorescence when these double strand segments melt. A melting curve isgenerally a plot of the fluorescence versus temperature of afluorescently stained nucleic acid. The shape of the curve containsinformation regarding the structure of the melting nucleic acid and maybe used as a specific, although low resolution, indicator of nucleicacid identity. This technique is widely used in real time PCR analysisand molecular biology.

In one aspect, MCA can be used to differentiate target nucleic acids ina mixture of a few different nucleic acids. In one aspect, nucleic acidlocalization and labeling is carried out as described above, with the MCdetection system modified to control and vary membrane filtertemperature or other factors known to cause nucleic acid melting. Thismay be accomplished by controlling the temperature of the fluid flowingthrough the microporous material, controlling the temperature of theentire disposable, solvent composition, etc. A nucleic acid countingmelting curve analysis (NAC-MCA) is performed by taking NAC measurementsat several microporous material temperatures and correlatingfluorescence changes versus temperature to known properties of thetarget nucleic acid.

For example, a target nucleic acid may have a melting point of 80° C.The detection instrument is set to make NAC measurements from about 75°C. to about 85° C. in 0.5° C. increments. Target nucleic acid willundergo a specific fluorescence reduction as the melting temperature isreached. Non target nucleic acids will most likely melt at a differenttemperature. The detection instrument is set to only count nucleic acidsthat melt within a predefined range. For applications involving limitednumbers of contaminating non target nucleic acid types, this addedspecificity can eliminate the complication of developing complimentaryprobes, stains, etc. It is contemplated that single strand RNA withextensive secondary structure (e.g., HIV, HCV, entrovirus, etc.) can beanalyzed by NAC-MCA, with the stained secondary structure melting at apredictable, unique temperature. Additionally, multiple complementarynucleic acid binding probes may be designed for a specific targetnucleic acid, for instance HIV, that all melt at a uniform temperature.In this case, localized HIV hybridized to multiple probes and stainedwith a suitable nucleic acid dye will melt at a well defined temperatureunique to the specific probe design.

In certain circumstances, localized nucleic acid morphology can be usedto improve detection SNR. As previously discussed, nucleic acids aremolecules of exceptionally high aspect ratio. Many target nucleic acidsare localized with clearly identifiable morphology. FIG. 11 shows humangenomic DNA localized on a nickel boron Anopore membrane stained withSyber Gold dye and imaged with the detector shown in FIG. 9 according toExample 12. The rod shaped DNA is clearly visible. In this case,although the pixel brightness of the DNA is greater than the surroundingbackground pixels, the DNA is identified primarily by shape. Imageanalysis techniques are available to aid in identifying particularshapes, correct for overlapping regions, improve image quality, etc. andimprove counting accuracy. Certain nucleic acids and staining techniquescan result in optically identifiable structures that do not easily fitwithin a compact detection zone, may present substantial image overlap,and otherwise require image analysis techniques to identify individualnucleic acids. These techniques are widely used and known to thoseskilled in the art.

iii. Counting

Immobilization of relatively pure analytes near the surface of themicroporous material, which is accomplished by the techniques describedherein, is an important variable with respect to counting the analyte.The term “counting” is defined herein as determining the number ofindividual analyte particles localized near the surface of themicroporous material at a particular area of the microporous material.Single molecule counting, even for relatively large, multilabeledanalytes, requires exceptional detectability. Consequently, singlemolecule counting requires immobilizing substantially purified analytesfree of competing or interfering substances. The following principlesdemonstrate how surface immobilization as accomplished by the techniquesdescribed herein enhance molecular counting.

For optical systems, it can be mathematically shown that the lightgathering ability of an instrument is proportional to the depth offocus. That is to say, detected fluorescent signals in an instrumentoptimized to interrogate fluorescent particles localized in a 1 microndeep region will be at least 100 times greater than an equally welloptimized instrument interrogating the same fluorescent particleslocalized in a 100 micron deep region. The 1 micron depth of focusinstrument could be used to stepwise interrogate the 100 micron deepregion, but would take 100 times as long.

Optical molecular counting requires isolating exceptionally smallvolumes for optical interrogation. The most straightforward approach tocounting the analyte is to interrogate a very small detection volume forthe presence or absence of a target molecule. As the detection volumebecomes larger, molecular resolution is lost, the background signalderived from nonspecific sources within the detection volume becomesgreater, and specific signal derived from the presence of the targetmolecule may be lowered, thereby lowering the practical ability todiscern when a target molecule is within the detection volume.

Although small detection volumes result in increased molecularresolution and improved optical signal to noise ratio, such volumes alsoaffect assay time. Given a detection volume (for instance based onrequired resolution and counting signal to noise ratio), the requiredcounting time is proportional to the total volume that must beinterrogated. For example, a 200 microliter (μl) patient sample maycontain only 10 target nucleic acid molecules. Assuming a detectionvolume of 10 cubic microns, a 200 microliter volume will have to beinterrogated 20 billion times to count the 10 target molecules. If the10 target molecules are localized to a surface 5 millimeters indiameter, the surface will have to be interrogated approximately 1million times. Localizing the target molecules to a filter surface asdisclosed herein represents an effective concentration improvement of20,000 based on detection, which translates to an equivalent savings inassay time.

Molecular location should be known during optical counting.Interrogating millions to billions of detection volumes within a samplecan take minutes to complete. Freely diffusing molecules can be countedmultiple times by high speed scanning detectors, or not at all withintegrating charge coupled device (CCD) array detectors. Thus, targetmolecule location is most easily controlled by immobilization near thesurface of the microporous material. In the case of nucleic acidcounting, it is preferable that the microporous material benonfluorescent and able to immobilize target nucleic acids underconditions where hybridization and/or fluorescent staining can occur.

For an acceptable analyte counting assay, the following conditionsshould be considered:

1. Signal-to-Noise Ratio (SNR): target analytes should be sufficientlyfluorescent to achieve an instrument signal-to-noise ratio consistentwith acceptable error rates. In the case of nucleic acids, calculationsshow a SNR of greater than 3 or from about 3 to about 50, from about 3to about 40, from about 3 to about 30, from about 3 to about 20, or fromabout 3 to about 10 is desirable for unambiguous, low error rate nucleicacid counting.

2. Assay Specificity: non target analytes should be nonfluorescentconsistent with acceptable assay specificity requirements.

3. Dynamic Range: target analytes should be localized near the surfaceof the microporous material as resolvable, discrete fluorescententities. That is to say, each target analyte should be individuallycountable from all others. Thus, each pixel or detection zone that thedetector interrogates should contain either 1 or 0 fluorescent targetanalytes, or signal processing technologies may be employed to identifythe boundaries of individual molecules to prevent miscounting.

The ratio of detected signal when a target analyte is present in theinterrogation volume to the detected signal in the absence of a targetanalyte is defined as the signal-to-noise ratio (SNR). The detectedsignal can be based simply on intensity at a certain wavelength, or acomplex parameter involving intensity, multiple wavelengths, phase,timing, etc. The required SNR is a function of instrumentation designand assay requirements. Example 14 below presents calculations for ascanning confocal epifluorometer based nucleic acid counter, includingestimates of required SNR, design interrogation area, required scantimes, and hardware assumptions.

When the analyte is a nucleic acid, a limiting factor for achieving highSNR in a nucleic acid counting assay is nonspecific tracer binding.Tracers, whether nucleic acid stains or oligo-based fluorescent probes,will likely be present near the surface of the microporous materialunassociated with target nucleic acids. This nonspecific tracer willgenerate a fluorescent signal when interrogated that must not bemiscounted as target nucleic acid.

For example, when localized target nucleic acids are to be labeled withan oligonucleotide based fluorescent probe, if each localized targetnucleic acid hybridizes with only one fluorescent probe in the detectionregion, and the relative fluorescence characteristics (quantum yield,wavelength, etc.) are the same for specific and non specifically boundprobes, the target nucleic acid is indistinguishable from randomlylocated nonspecifically bound probes. This is true regardless of thedegree of fluorescent labeling on the probe, which can includefluorescent bead labels, quantum dots, surface plasmon resonanceparticles, multiple labels, etc. In this case, a true SNR of 10 requiresa minimum of 10 probes specifically bound to the target nucleic acid inone detection region, assuming the probe nonspecific binding (NSB)remains as isolated, individual molecules that are not clumped together.

The above situation is based on simple intensity measurements ofdetection zone fluorescence employing multiple probes each labeled witha common fluorescent tag. If each probe is labeled with a clearlyseparate fluorescent tag to form a separate, identifiable detectabletracer, higher effective SNR at lower probe numbers may be possible. Forexample, the required SNR for a counting assay is based on the allowablefrequency of miscounting a non specific event. In a simplified case,this is similar to determining the probability a non specific event willbe identical (as detected by the instrument detector) to the desiredspecific event. Suppose a common tag (for instance intensity measurementof one fluorescent wavelength) system operates with 5 probes. Theprobability a non specific event will be as intense as five combinedprobes is low. A system based on five probes with separatelyidentifiable tags (for instance wavelength and intensity) has an evenlower probability a non specific event will be as intense and containall the required wavelengths in the correct ratios. Accordingly, morecomplex detection schemes may be employed to maintain high effective SNRwith reduced probe numbers.

High effective SNR may also be achieved with lower probe numbers byemploying techniques where non specifically bound probes do not elicitthe same optical response as those specifically bound to a targetnucleic acid. This is similar to the use of nucleic acid stains thatundergo substantial quantum yield increase on binding to nucleic acids.Enzymes are known to frequently lose substantial activity when incontact with surfaces. As disclosed herein, specifically bound probeslabeled with enzymatic tags may be considered suspended over themicroporous surface on the localized nucleic acid rather than in directsurface contact as are the non specifically bound probes. Accordingly,enzyme labeled probes may exhibit high effective SNR with low probenumbers.

iv. Correlating the Analyte Concentration

The term “correlating” as used herein is defined as quantifying theconcentration of the analyte present in a known volume of sample basedon the number of analyte particles counted on the microporous materialand corrected for known relationships such as the localizationefficiency of the microporous material, labeling efficiency, detectionefficiency, field of view, etc.

In one aspect, qualitative nucleic acid counting or detection may beadvantageously performed. In this aspect, final correlation of detectedsignal to the initial sample concentration is of lesser importance. Forinstance, identification of the presence or absence of a pathogen,rather than exact concentration, is frequently sufficient for diagnosis.Examples include, but are not limited to, identification of biologicalwarfare agents, identification of Strep A bacteria in throat swabs,identification of the MecA gene in S. Aureus cultures to determine drugresistance, etc. The microporous materials, methods, and articlesdescribed herein provide qualitative analysis through speed, ease ofuse, elimination of molecular amplification steps (such as PCR),simplified hardware requirements, the ability to use multiple sampletypes without preprocessing, etc. without consideration of final resultcorrelation to initial concentration.

In another aspect, the ability to correlate the counted nucleic acidswith an initial sample nucleic acid concentration is important toquantitative analysis. Indeed, methods of counting immobilized nucleicacids have existed for many years. For instance, it is well known in theart nucleic acids may be immobilized to quartz surfaces by simplycontacting a relatively high concentration sample with a clean surface.Some of the nucleic acids will “stick” to the quartz and may be countedby Atomic Force Microscopy (AFM), Scanning Electron Microscopy (SEM),etc. In one aspect, the microporous materials, methods, and articlesdescribed herein improve the current state of the art by the ease andspeed with which nucleic acids may be localized to the microporousmaterial, the wide variety of sample types and purity allowed forlocalization to the microporous material, and the ability to correlatethe counted nucleic acids to the initial nucleic acid sampleconcentration.

The microporous materials described herein permit the localization ofanalytes through a process similar to mechanical filtration. Thislocalization step is essentially quantitative for certain analytes suchas certain sizes of nucleic acids, with nearly 100% of all targetnucleic acids localized near the surface of the material foridentification by detection such as counting or additional processingsteps such as PCR, etc. This very high localization efficiency permitsthe analysis of very low concentration substances that may only occur asa few copies per milliliter of sample.

The repeatability of localization also effects correlation. For example,if the microporous material always captures 2% of the nucleic acids, acorrelation between nucleic acid count and initial sample concentrationmay be established, although certainly with lower sensitivity than if100% of target nucleic acids are always captured and available foranalysis. The microporous materials, methods, and articles describedherein permit localization that is both exceptionally efficient andrepeatable.

Instrumentation, hardware, and processing parameters should bepredictable for successful correlation. For example, if a quantitativeassay for a circulating virion is desired, the following parametersshould be known in order to correlate the final detected nucleic acidcount with initial virion concentration.

1. Virion lysis efficiency

2. Initial sample volume across the capture membrane

3. Nucleic acid capture efficiency

4. Nucleic acid labeling efficiency

5. Labeled nucleic acid detection efficiency

6. Percent of membrane interrogated by detector

7. System linearity versus detected signal

Unlike existing analyte counting methods, correlation between finalanalyte count and initial sample volume through efficiency andrepeatability of the above defined parameters is improved substantially.FIGS. 12 a-12 h depict processed, negative images of the YOYO-1 labeledcalf thymus DNA that was optically detected according to Example 13.Relative DNA concentrations in the filtered test samples were increasedfrom 1 in FIG. 12 a up to 128 in FIG. 12 h. As seen in FIGS. 12 a-12 h,individual nucleic acids can be easily counted in the range of interestfor high sensitivity diagnostic assays.

FIG. 13 is a graph of uncorrected nucleic acid count vs. nucleic acidconcentration, showing a curve of the counted calf thymus DNA labeledwith YOYO-1 that was localized to the Anopore membrane surface from aninitial sample volume of 100 microliters. The curve count valuescorrespond to the processed images depicted in FIGS. 12 a-12 h. Thegraph of FIG. 13 is an example of an uncorrected NAC assay calibrationcurve using the techniques described herein.

FIG. 14 is a graph showing the corrected nucleic acid count with respectto nucleic acid concentration (i.e., corrected sample concentrationderived from images vs. true relative sample concentration) derived fromFIG. 13 and corrected for detector and immobilization efficiency.

D. Destabilization and Manipulation of Localized Analytes

In one aspect, once the analyte is localized on the microporous materialor substrate, the localized analyte can be destabilized for furthermanipulation. The term “destabilized” as used herein is defined as anyprocess that weakens the interaction between the analyte and the surfaceof the microporous material or substrate so that the localized analytebecomes more accessible for chemical manipulation. Typical interactionsthat can occur between the analyte and the microporous material orsubstrate include, but are not limited to, ionic or charged-chargedinteractions, hydrophobic/hydrophilic interactions, andmechanical/physical entanglement of one or more analytes. Varyingdegrees of destabilization of the analyte are possible, and will varydepending upon the analyte, the microporous material/substrate, anddestabilizing technique that is employed. For example, destabilizationof the analyte can completely remove the localized analyte from themicroporous material and solubilize the analyte. Alternatively, aportion of the analyte can be destabilized while another portion of theanalyte can interact with the microporous material.

In one aspect, described herein are methods for detecting an analyteinvolving the steps

-   (a) passing a fluid sample containing the analyte through or into a    microporous material, wherein the analyte is localized near the    surface of the microporous material;-   (b) destabilizing at least some of the localized analyte; and-   (c) detecting the destabilized analyte.    Any of the analytes, microporous materials, suspension matrices, and    detection techniques described above can be used in this aspect.

In one aspect, the destabilizing step includes (1) contacting thelocalized analyte with a base; (2) agitating the localized analyte; (3)heating the localized analyte; (4) contacting the localized analyte withone or more ionic species; (5) contacting the localized analyte with oneor more enzymes; (6) applying an electrical charge or ionizing energy tothe localized analyte; (7) contacting the localized analyte with anorganic solvent, or a combination of any of the steps above. It iscontemplated that the destabilization step can involve one of thetechniques described above or two or more techniques that are performedconcurrently/simultaneously or sequentially.

In one aspect, the destabilizing step involves contacting the localizedanalyte with a base. In one aspect, the base has a pH of greater than 9.In another aspect, the base has a pH of 9 to 14, 9 to 13, 9 to 12, or 10to 12. Any base known in the art can be used in this aspect. Examples ofbases include, but are not limited to, an inorganic base or a buffer. Inone aspect, the base can be NaOH, tris, carbonate/bicarbonate, and thelike.

In another aspect, the destabilizing step involves agitating thelocalized analyte. The term “agitating” as defined herein includes anytechnique for destabilizing the localized analyte by physical force.Examples of techniques useful for agitating the localized analyteinclude, but are not limited to, sonication, vortex mixing, or pumping.

In a further aspect, the destabilizing step heating the localizedanalyte above 25° C. In another aspect, the localized analyte is heatedfrom 60° C. to 100° C. In one aspect, the microporous material orsubstrate containing the localized analyte is submersed in a solutionfollowed by heating the solution.

In another aspect, the destabilizing step involves contacting thelocalized analyte with one or more ionic species. Any ionic speciesknown in the art can be used herein. Depending upon the analyte to bedestabilized, the ionic species can be positively or negatively charged.Not wishing to be bound by theory, it is believed that the ionic speciescompete with the localized analyte for binding sites present on themicroporous material or substrate. In one aspect, when the analyte is anucleic acid, examples of ionic species useful herein include, but arenot limited to, phosphate ions, borate ions, or a combination thereof.

In another aspect, the destabilizing step involves contacting thelocalized analyte with one or more enzymes. The selection of the enzymewill vary depending upon the localized analyte. In one aspect, when theanalyte is a nucleic acid, the enzyme can be a restriction endonuclease,a nick enzyme, or a helicase.

Other methods for destabilizing a localized analyte involve contactingthe localized analyte with an organic solvent that can solubilize aportion or all of the analyte. For example, a hydrocarbon solvent can beused to destabilize a hydrophobic analyte. In another aspect, when asuspension matrix is localized on the microporous material or substrate,an organic solvent can destabilize the suspension matrix as well.

Alternatively, in another aspect, an electric current can be applied toa microporous material or substrate containing the localized analyte inorder to destabilize the analyte. For example, electrodes can beattached to the microporous membrane and an electrical charge can beapplied. In another aspect, ionization energy can be used to delocalizethe analyte. The amount of charge or energy that is applied can varydepending upon the localized analyte. By varying the amount of currentor energy, it is contemplated that the degree of destabilization can bevaried as well.

Once the analyte has been destabilized, the destabilized analyte can bechemically manipulated using any of the techniques described above. Inone aspect, when the destabilized analyte is a nucleic acid, thedestabilized nucleic acid can be detected, isolated, purified, and/oridentified using any of the techniques described above. In one aspect,the destabilized analyte can be detected by amplification andhybridization. It is believed that after the destabilization step, thedestabilized analyte is more accessible for manipulation. Thus, thedetection and identification of the analyte such as a nucleic acid canbe greatly enhanced compared to an analyte that is localized and notdestabilized.

In one aspect, when the analyte is a nucleic acid, the detection of thedestabilized nucleic acid by PCR amplification is from 5 to 75%, 10 to60%, or 20 to 50% higher than the localized nucleic acid. For example,PCR amplification of localized nucleic acids is approximately 1-5% asefficient as fully dissolved nucleic acid, but this figure jumps toapproximately 10-20% when destabilization reagents composed of, forexample, Tris(tris (hydroxymethylaminomethane) (pH 11) are used. Notwishing to be bound by theory, it is believed the localized nucleic acidis not being eluted from the membrane surface into solution directly bythe destabilization reagent, but can improve PCR amplificationefficiency by either allowing the localized nucleic acid to entersolution under PCR thermal cycling conditions or simply improvingreagent access to the localized nucleic acid.

In another aspect, mild ultrasonic mixing of destabilization reagentresults in the PCR amplification efficiency jumping from approximately10% (without sonication) to over 50% with sonication. In another aspect,the combination of pH and ultrasonic mixing causes a measurable amountof localized nucleic acid to destabilize.

In one aspect, a suspension matrix is localized on the surface of themicroporous material prior to contacting the microporous material withthe analyte. In this aspect, the analyte can then be localized on thesuspension matrix. Upon destabilization, the suspension matrix and/orthe localized analyte can be destabilized. In one aspect, when DNA isused as the suspension matrix, the matrix can be destabilized forimproved detection. Alternatively, the suspension matrix can be mixedwith the analyte prior to localization on the microporous substrate.

In another aspect, a method for detecting a nucleic acid involves

(a) contacting a fluid sample comprising the nucleic acid with asurface, wherein the nucleic acid is localized near the surface;(b) destabilizing at least some of the localized nucleic acid; and(c) detecting the destabilized nucleic acid.The surface in this aspect, the surface can be any material that canform an interaction with the analyte that is capable of beingdestabilized using the methods described herein. Examples of surfacesinclude, but are not limited to, glass, plastic, metals, ceramics, amicroporous material described herein, and the like.

E. Articles and Kits

Any of the microporous materials and composites described herein can bepart of a filtration device. The phrase “filtration device” as referredto herein is defined as any device that contains at least onemicroporous material described herein.

In one aspect, a filtration device is depicted in FIG. 15 a. A filterassembly 1530 includes a well 1532 for holding a test sample 1533containing an analyte. While only one well is shown in FIG. 15 a, itshould be understood that a plurality of wells can be used that are partof a filter plate 1534, which can have a multiwell format, such as a 96or 384 well filter plate. A bottom opening 1538 of well 1532 is coveredwith a microporous material 1540 for analyte localization. In oneaspect, a 0.2 micron Anopore membrane filter can be heat fused to abottom portion of well 1532. The bottom opening 1538 is in fluidcommunication with a vacuum source (not shown), which can provide adifferential of about 5-10 psi to aid in filtering test sample 1533through analysis filter 1540. In addition, analysis filter 1540 may beconfigured for optical molecular detection of analyte. For example,analysis filter 1540 can be removably attached to a bottom portion ofwell 1532, to allow removal of filter 1540 for use in an opticaldetection system. Additionally, filter assembly 1530 may be designed, inconjunction with a suitable detector, to permit optical moleculardetection on analysis filter 1540 without removal.

In another aspect, FIG. 15 b is a schematic depiction of a filter systemthat employs filter assembly 1530 with prefiltration according to analternative embodiment. As depicted in FIG. 15 b, filter assembly 1530can be used in conjunction with a prefiltration device 1535, such as asyringe type filter (shown) or filter plate (fixed or removable, notshown). The prefiltration device 1535 contains a prefilter 1536 that isconfigured to retain contaminants or impurities larger than themicropores in prefilter 1536, while allowing particles containinganalytes in the fluid to pass through the first filter. In one aspect,when the analyte is a nucleic acid, the nucleic acid can includevirions, bacteria, spores, and the like. The prefilter 1536 can be asurface filter such as a membrane filter, or prefilter 1536 can be adepth filter.

During use, test sample 1533 is filtered through prefilter 1536 intowell 1532. Additional reagents, such as virion lysing agents, dyes,buffers, etc., may be added to the filtered test sample in well 1532.The filtered test sample is incubated and then filtered through analysisfilter 1540 to effect nucleic acid capture and localization.

In one aspect, filter assembly 1530 is a rapid viral load assay, such asin testing for HIV, hepatitis C, or other infectious diseases. In thisaspect, only the infectious virion particles are to be counted. That isto say, many virus-like particles are empty of nucleic acid and arenoninfectious. Likewise, freely circulating viral nucleic acid(unencapsulated) is also noninfectious. In using the filter system shownin FIG. 15 b, intact, unlysed virions will pass through prefilter 1536,while larger impurities and free nucleic acids will be retained. Thefiltrate virions in well 1532 are then treated to release nucleic acidsfor capture on analysis filter 1540, while proteins, viral capsuledebris, other small impurities, etc. will pass through filter 1540. Theonly nucleic acids captured on analysis filter 1540 are those that werecontained within intact virions. This way, only infectious virions arecounted.

For example, an analysis of HIV viral load may be performed with filterassembly 1530 as part of a high sensitivity assay that requiresapproximately 200 μl patient samples to capture sufficient nucleic acidsto be statistically meaningful. In order to obtain such a sample, a 200μl human plasma sample is prefiltered using prefilter 1536, as shown inFIG. 15 b, to remove large impurities and free nucleic acids. An intactHIV virion is known to be approximately 0.1 microns in diameter and willpass through a 0.2 micron prefilter. The prefiltered sample is thenprocessed in well 1532 to lyse the virions to release viral RNA, and theviral RNA is then captured on analysis filter 1540 according to thedisclosed methods. Alternatively, the prefiltered sample can bedeposited into a sterile, clean container (not shown), and thenaccurately pipetted along with lysing reagents, etc. into well 1532. Theviral RNA localized onto the surface of filter 1540 can then besubjected to analysis such as optical molecular counting or conventionalPCR type analysis.

In one aspect, the filtration device is composed of a well body havingan inlet and an outlet, wherein the well body has an inner wall andouter wall, and (b) a filter composed of any of the microporousmaterials including composites and modified-microporous materialsdescribed herein, wherein the filter is attached to the inner wall ofthe well body. In one aspect, the filter can be attached to the innersurface of the well body directly with an adhesive. In another aspect,the filter can be attached to the inner surface of the well body by withthe use of a filter holder. In one aspect, the well body is composed ofany material that is chemically inert and that can be subjected to heatwithout altering the dimensions or structure of the well body. In oneaspect, the well body is composed of a plastic such as polypropylene.

Once the sample containing the analyte is passed through the inlet ofthe well body, contacted with the filter and the analyte is localizednear the surface of the microporous material, and the remaining solutionis passed through the filter and exits the outlet, the localized analytecan be further manipulated. In one aspect, the inlet and outlet of thewell body can be receptive to a cap or seal. For example, the inletand/or outlet can be threaded to receive a screw-on cap. Alternatively,the inlet and/or outlet can contain edges or contours that permit a capto be snapped on the inlet or outlet. In one aspect, the cap or seal canbe any of the materials that the well body is composed of.

Several aspects of the filtration device described above are depicted inFIG. 16. In one aspect, FIG. 16 a depicts a filter assembly, generallydesignated as 1650, designed to fit within a commercially availablethermal cycler 1670. The filtration device is composed of well body1651, filter holder 1652, and outlet cap 1653. Analysis filter 1654 isattached to filter holder 1652 by heat fusing or other means to ensuresample 1655 is filtered through analysis filter 1654 during operation.During assembly, filter holder 1652, including attached analysis filter1654, is placed within well body 1651 and secured by mechanicalinterference between tapered surfaces generally shown as 1656.Alternatively, these surfaces may be adhesively bonded or otherwiseaffixed in a leak free manner. Clearance 1657 between well body 1651 andfilter holder 1652 is allowed so as not impose undue stress on attachedanalysis filter 1654, but must be minimized to prevent samplecontamination and carryover. During use, as shown in FIG. 16 b, sample1655 is placed in filter assembly 1650 and outlet cap 1653 has not beeninstalled. Mild vacuum 1666, pressure, or centrifugal force is used toeffect fluid flow of sample 1655 through analysis filter 1654. Filtrate1658 flows from filter assembly 1650 to waste (not shown) through outlet1660 while nucleic acids 1659 are retained on analysis filter 1654 forfurther processing. Localized analytes 1659 and analysis filter 1654 maybe rinsed or otherwise processed at this point to minimize effects ofNSB, carryover, etc. The outlet 1660 of relatively dry filter assembly1650 containing analytes 1659 on analysis filter 1654 is capped withoutlet cap 1653 (FIG. 16 a) to prevent further fluid flow from theassembly during further processing. The fluid seal for outlet 1660 isaffected by mechanical interference between tapered surfaces generallyshown as 1661 present on outlet 1660 and outlet cap 1653. Other methodsof sealing outlet 1660 known to those skilled in the art are alsocontemplated, such as valves, plugs, etc.

FIG. 16 c shows filter assembly 1650 configured for further processing.For example, reaction reagents 1662 such as PCR master mix orhybridization reagents for nucleic acid labeling have been added and arein contact with nucleic acids 1659 on analysis filter 1654. Cap 1663 hasbeen added to seal the top opening 1664 of filter assembly 1650.Alternatively, top opening 1664 may be sealed with film as is known tothose skilled in the art. In this configuration, the assembly may besubjected to thermal cycling or other processes as required forconventional PCR, RT-PCR, hybridization, etc. processes for analysis.Additionally, this embodiment may be useful for other detection schemessuch as molecular counting. In this case, well body 1651 may be shorterto accommodate certain detector optical designs. In all cases, however,the ability to retain liquids in contact with the membrane duringprocessing by reversibly capping openings is important.

Another filter assembly is shown in FIGS. 17 a and 17 b. This filterassembly, generally designated 1770, is designed to operate with flatsurface heat transfer for thermal cycling rather than the well typeformat described for the filter assembly shown in FIG. 16. In oneaspect, the microporous material is composed of aluminum oxide, whichhas excellent heat transfer properties compared to the walls of aplastic well as found on most PCR thermal cyclers. Accordingly, thermalcycling time improvements may be realized by providing a filter assemblyand thermal cycler designed to transfer heat across the analysismembrane.

In another aspect, the filtration device contains two or more wellbodies, wherein each well body has a top opening and a bottom opening,and (b) a filter composed of any microporous material described herein,including composites and modified-microporous materials, wherein thefilter covers the bottom opening of each well body. FIG. 17 depictscertain aspects of this embodiment. The well body can be composed of anyof the material described above.

FIG. 17 a shows filter assembly 1770 during sample preparation installedin vacuum filtration device 1771. Alternatively, any device thatfacilitates the movement of fluid through the filter of the filtrationdevice can be used in this embodiment. Examples of such devices include,but are not limited to, filtration by pressure, centrifugation, orfluidic pumping. Filter assembly 1770 is shown as a three well assembly,but generally may contain any number of wells. For instance, currentlyused multiwell plates frequently contain 96 or 384 separate wells.Filter assembly 1770 contains analysis filters 1772 attached by heatsealing or other means so as to allow for analyte localization to thefilter surface during filtration as previously described. Solutions tobe analyzed, in this case whole blood lysate 1773, are contained in thewells of filter assembly 1770. During vacuum filtration, a vacuum sealis established at vacuum sealing surface 1775. Filter assembly 1770 isshown with a compliant support plate 1774 that allows the assembly todeform under vacuum to affect a more uniform vacuum seal on vacuumsealing surface 1775. In one aspect, the vacuum causes whole bloodlysate 1773 to filter through analysis filters 1772. In this aspect,nucleic acids present within the whole blood lysate 1773 are localizedon the microporous material 1772, while whole blood filtrate 1776traverses the analysis filters 1772 and eventually to waste (Not shown).As described in the previous examples, the analysis membranes 1772 maybe rinsed prior to further processing.

FIG. 17 b shows filter assembly 1770 during RT-PCR processing. In thisaspect, prior to processing, the disposable wells can be sealed toprevent leakage, contamination, and evaporation. The analysis filter1772 can be sealed as shown with a sheet of thin (1 mil) plastic, eitherby adhesive bonding (sticky film) or by heat sealing to the membrane orthe housing around the membrane periphery as shown as membrane seal1780. This membrane should be thin to allow rapid heat transfer throughthe combined sealing/filtration membranes. In one aspect, heat transferanalysis indicates the thermal resistance of the Anopore filter isnegligible compared to typical sealing films. Nevertheless, evenemploying conventional plastic sealing materials (polypropylene films,etc), this thermal resistance is less than the glass capillaries used inthe Lightcycler RT-PCR instrument (Roche), and many times less thanplastic well based thermal cyclers. Prior to filtration membranesealing, an optional clean blotting step can be performed to remove anyhanging filtrate drops or liquid. This may be advantageouslyaccomplished with disposable blotting material (not shown), where thefiltration assembly 1770 is removed from the vacuum filtration device1771, quickly blotted against a disposable blotting surface (forinstance blotting paper) then membrane sealed either with adhesive orthermal sealing membranes.

In this aspect, the top opening of the disposable wells can be sealed.Additionally, sealing should be accomplished so as to allow opticaldetection of the reactions. A top seal plate 1786 is shown in FIG. 17 bas a separate molded piece containing an integral thin optical window1782 for optical analysis. Although shown in gray with transparentwindows, the top seal plate can be composed of a clear low fluorescentmaterial with integral windows. As shown, the top seal plate 1786 formsa seal 1783 with filter assembly 1770 within the well just above the PCRreaction 1788 liquid level. During thermal cycling, PCR reaction 1788solution temperature must be homogenous (isothermal), liquid evaporationmust be minimized to maintain consistent reaction conditions, andcondensation should be controlled on the optical window 1782 to allowaccurate optical measurements. Accordingly, the distance between the PCRreaction 1788 liquid surface and optical window of the seal plate 1786should be minimized consistent with ease of use, materials ofconstruction, and desired heat and mass transfer conditions of theoptical window to minimize condensation.

Referring to FIG. 17 b, thermal cycling is accomplished by directconductive heat transfer through the membrane seal 1780/analysis filter1772 from a thermoelectric module 1785 operated in a temperature sensingfeedback loop. That is to say, a thermoelectric module 1785 (TEM,peltier heater/cooler) is in intimate contact with the membrane seal1780 with no intervening circulating air or water baths, dead air gapsassociated with slip fits into aluminum block heaters, etc. Thermalcontact is improved by using a compliant multiwell plate design, in thatthe compliant support plate 1774 of filter assembly 1770 has sufficientdeformability to allow the bottom membrane seal 1780 to conform to theflat surface of the thermoelectric heating/cooling module 1785. In thisway, excellent thermal performance with better accuracy and speedcompared with existing thermal cycling equipment is anticipated.

An embodiment of FIG. 17 b is shown in FIGS. 18 and 19. In theseembodiments, filtration device comprises (a) a plate having a firstsurface and a second surface, wherein the plate has at least one hole,wherein the hole has a fixed width at the first surface and the secondsurface of the plate, and (b) a microporous material having a firstsurface and a second surface, wherein the first surface of themicroporous material is adjacent to the second surface of the plate,wherein the microporous material covers each hole in the plate. The term“adjacent” as referred to herein is defined as the plate and anymicroporous material described herein that are in physical contact withone another. The term “adjacent” also refers to the plate andmicroporous material that is separated by another material. For example,a prefilter can be placed between the plate and the microporousmaterial.

In FIG. 18, plate 1800 is composed of any material that possessesrigidity, low fluorescence, ease of manufacture, and thermalconductivity. In one aspect, the plate is composed of anodized aluminumor plastic. In FIG. 18 a, 12 reaction wells 1801 have been formedthrough plate 1800; however, any number of holes, including only onehole, can be present in the plate. In one aspect, Anopore membrane 1802has been bonded to the bottom surface of plate 1800, thereby forming thebottom of the reaction wells 1801. The membrane may be bonded withliquid adhesives such as epoxy, acrylic, etc. or with adhesive tapes.The microporous material covers each hole in the plate. Referring toFIG. 18 b, the wells may have a larger top portion 1804 and a smallerbottom portion 1805 to aid in optical detection, sealing, liquidhandling, etc. In one aspect, the larger top portion 1804 can receive aprefilter that can remove impurities. An analyte containing sample isfiltered and rinsed through the microporous material, thereby localizingthe analyte for further processing and detection. In one aspect,referring to FIG. 19 a, when the analyte is a nucleic acid, the driedbottom surface of the microporous material 1902 containing localizedanalyte can be sealed by heat welding polypropylene heat sealing film1910 directly onto the dry membrane. Reaction liquid 1913 such as, forexample, PCR master mix, can then added into wells 1901. The tops ofwells 1901, containing the reaction liquid 1913 can then be sealed byclear adhesive tape 1911 (FIG. 19 b) or clear snap-on top 1912 (FIG. 19a). This assembly can then processed as described in, for example, FIG.17.

In another aspect, the microporous material used for concentration andlocalization of nucleic analytes can be part of a cascade filterassembly 2080 in FIG. 20. The filter assembly 2080 includes a removablehousing 2082 for receiving a sample containing the analyte. A firstportion 2083 of housing 2082 is configured to contain a first filter2084, such as a 0.2 micron Anopore membrane filter for prefiltering thetest sample. A second portion 2085 of housing 2082 is configured tocontain a second filter 2086 for analyte localization, such as a 0.2micron Anopore membrane filter. The filters 2084 and 2086 are arrangedin an in-line filter type configuration within housing 2082 such thatfilter 2084 is adjacent to and precedes filter 2086. A sample entrancetube 2087 is in fluid communication with a first chamber 2088 in firstportion 2083, and an exit tube 2089 is in fluid communication with asecond chamber 2090 in second portion 2085 of housing 2082.

In one aspect, during use of filter assembly 2080, a preprocessedpatient sample such as a serum sample is deposited through tube 2087into chamber 2088 and onto filter 2084. As the sample flows throughfilter 2084 and then filter 2086, nucleic acids are localized to thesurface of filter 2086. As shown in FIG. 20, filter 2086 including thecaptured nucleic acids can then be physically removed from housing 2082for further processing and/or optical nucleic acid counting such asdescribed hereafter.

In a further embodiment, the membrane filter used for concentration andlocalization of analytes can be part of a flowcell filter assembly 2100as depicted in FIG. 21. The flowcell filter assembly 2100 includes ahousing 2102 for receiving a sample or other sample containing theanalyte. The housing 2102 is configured to hold an analysis filter 2104.In one aspect, the analysis filter 2104 is a 0.2 micron Anopore membranefilter for nucleic acid localization. The filter 2104 rests on anoptional porous filter support 2105. If filter 2104 has a sufficientlysmall diameter, then porous filter support 2105 may be omitted. Anentrance tube port 2106 is in fluid communication with a first chamber2107 adjacent to filter 2104, and an exit tube port 2108 is in fluidcommunication with a second chamber 2109 adjacent to filter support 2105in housing 2102. An optical window 2110 is located on a surface ofhousing 2102 to allow transmission of light 2112 to and from a detector(not shown) to the surface of filter 2104. The light 2112 is operativelyconnected to a detector such as a fluorometer (not shown).

During use of flowcell filter assembly 2100, a sample is injectedthrough tube port 2106 into chamber 2107 and onto filter 2104. As thesample flows through filter 2104, analytes are localized on the surfaceof filter 2104. In one aspect, when the analyte is a nucleic acid, thenucleic acids in the sample are labeled prior to injection of the sampleinto flowcell filter assembly 2100. However, the nucleic acids can belabeled after localization to the surface of filter 2104. In thisaspect, the nucleic acids on filter 2104 may be counted in flowcellfilter assembly 2100 through optical window 2110 by use of a fluorometersimultaneously with sample fluid flow and filter membrane processing.This potentially improves nucleic acid detectability thru betterbackground signal rejection. Sample prefiltering, if required, is doneprior to injection of the sample into flowcell filter assembly 2100.Ports 2106 and 2108 may be capped or otherwise constricted to allowfurther processing of the localized nucleic acids, such ashybridization, PCR reactions, etc. to occur.

FIG. 22 shows a modification of a generalized well type format thatprovides for particulate prefiltration immediately prior to analytelocalization. In some cases, environmental dust, mold, bacteria, etc.may settle into the well containing the analyte sample to be analyzedand create erroneous signals for analyte counting. A filter assembly tominimize this effect is designated generally as 2200. The filterassembly 2200 has a housing defining a first chamber 2201 having anopening 2203, which allows deposition of an analyte sample 2202 withinchamber 2201. The bottom of chamber 2201 contains a prefilter 2204,which is selected so as to retain particles larger than about 0.2microns and pass the analytes. Below prefilter 2204 is a fluidpassageway 2205 that conveys a prefiltered analyte sample to an analysisfilter 2207, which is disposed in the housing at one end of a secondchamber 2209. At least part of one surface of passageway 2205 is definedby an optical window 2206, which allows the surface of analysis filter2207 in fluid communication with passageway 2205 to be detected.

During use of filter assembly 2200, analytes contained within sample2202 are localized on analysis filter 2207 as the sample flows thrufilter 2207 to form liquid waste 2208 located in chamber 2209. A ventport 2210 may be connected to a vacuum source to aid and control fluidflow, or chamber 2201 may be slightly pressurized as is well known tothose skilled in the art. The waste 2208 may be contained within chamber2209 as shown, or may be removed from chamber 2209 to a central wastecontainer (not shown). The filter assembly 2200 may be used withadditional prefiltration as described herein to remove intrinsic nucleicacids, particles, and other impurities prior to sample processing (e.g.,virion lysis, etc.) in chamber 2201. The filter 2204 simply removespotentially interfering particulate immediately prior to nucleic acidimmobilization on filter 2207.

FIGS. 23 a and 23 b depict a flowcell device for analyte counting withgreatly reduced diffusion distance, which can be used with solid surface(non-filter) localization. Low concentrations of analytes requireconsiderable time to diffuse to solid surfaces. FIG. 23 a shows a planview and FIG. 23 b shows a side view of a solid surface localizationflowcell device 2300. The flowcell device 2300 has an inlet tube 2301for introduction of an analyte containing sample 2302 into an inletchamber 2307 defined by a first portion of a housing. At least part ofone wall of chamber 2307 is formed by a flow distribution structure2303, which is between chamber 2307 and a localization passageway 2304defined by a second portion of the housing. Referring to FIG. 23 b, atleast one of the major surfaces of the second portion of the housingdefining localization passageway 2304 is composed of an optical window2305. In addition, the interior of at least one of these major surfaceshas been treated to localize the analyte from the sample as alocalization surface 2306. The localization surface 2306 can be onoptical window 2305, on a surface opposite optical window 2305, or onboth.

The distribution structure 2303 is generally composed of a porousmaterial providing sufficient pressure drop to cause substantiallyuniform flow downstream in localization passageway 2304. Under certaincircumstances, flow distribution structure 2303 may be configured as anarrow opening into localization passageway 2304. The localizationpassageway 2304 is generally of flat construction with dimensions ofabout 1 to about 10 mm width and length, and a thickness of about 10 toabout 50 microns. That is to say, the analyte containing sample 2302flows through a passage about 1 to about 10 mm wide, about 10 to about50 microns high, and about 1 to about 10 mm long.

During operation of flowcell device 2300, the analyte containing sampleflows into the device, where it is uniformly introduced intolocalization passageway 2304. As the sample slowly flows through thispassageway, analytes are localized onto localization surface 2306relatively rapidly due to the very small diffusion distance (about 10-50microns) afforded by the passageway height. The remaining fluid passesout of localization passageway 2304 as waste. Localized analyte is thenoptically detected on surface 2306. This flowcell device 2300 can beconfigured with gravity, pressure, or vacuum flow control, and may bepart of a disposable device. Additionally, flowcell device 2300 can beconfigured to process samples remotely from an optical detector for anextended period. Such samples are then quickly optically analyzed afterlocalization is complete.

Any of the articles and filtration devices described above can beincorporated into a kit. The kit also includes any of the detectionmeans described above for detecting the localized analyte. In oneaspect, an optical detector is used to detect a labeled nucleic acid.

F. General Terms

Before the present compositions, articles, devices, and/or methods aredisclosed and described, it is to be understood that they are notlimited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, and methods described and claimed herein aremade and evaluated, and are intended to be purely exemplary and are notintended to limit the scope of what the inventors regard as theirinvention. Efforts have been made to ensure accuracy with respect tonumbers (e.g., amounts, temperature, etc.) but some errors anddeviations should be accounted for. Unless indicated otherwise, partsare parts by weight, temperature is in ° C. or is at ambienttemperature, and pressure is at or near atmospheric. There are numerousvariations and combinations of reaction conditions, e.g., componentconcentrations, desired solvents, solvent mixtures, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the describedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

A. Materials

In a rapid viral load assay, nucleic acids tested may be RNA or DNAhaving about 8,000 bases or base pairs. Nucleic acid (DNA and RNA)ladders in this size range were tested for localization. Unlessotherwise specified, EcoR I digest of lambda phage DNA with fragments of3,530, 4,878, 5,643, 5,804, 7,421, and 21,226 base pairs was used as themodel nucleic acid. Comparison to other DNA and RNA ladders withfragments between 50 and 10,000 bases or base pairs indicated similaritybetween RNA and DNA localization. Gel electrophoresis on filterednucleic acid ladders indicated higher retention for molecules largerthan about 1,500 bases or base pairs. Purified Calf Thymus DNA of 13,000average base pair length was filtered under varying conditions and theresults noted in the Examples below. Filtration was done with a filterassembly including Millipore multiwell filter plates modified to include0.2 micron Anopore membrane filters similar to the well assembly shownin FIG. 15 a with modified and unmodified 0.2 micron Anopore membranefilter surfaces of approximately 5 millimeter diameter.

1. Example 1

FIGS. 24 a and 24 b are graphs of nucleic acid retention vs. NaClconcentration for plain unmodified, diol (acid hydrolyzedglycidoxypropyltrimethoxysilane) modified, andamine(aminopropyltrimethoxysilane) modified 0.2 micron Anoporemembranes. FIG. 24 a shows a 0 to 1000 mM NaCl range, while FIG. 24 bshows an expanded 0 to 100 mM range from FIG. 24 a. These results weregenerated in 1 mM Tris buffer, pH 8.0. As is apparent from the graphs,100 mM of salt is required for substantially complete nucleic acidretention on the plain and diol modified membranes, with substantiallyno salt effect noted for the amine modified membrane.

2. Example 2

FIGS. 25 a and 25 b are graphs of nucleic acid retention vs. saltconcentration (chaotropic and NaCl) for plain unmodified Anoporemembranes. FIG. 25 a shows a 0 to 4500 mM salt range, while FIG. 23 bshows an expanded 0 to 100 mM range from FIG. 25 a. These results weregenerated in 1 mM Tris buffer, pH 8.0, using the DNA digest cited above.As indicated in the graphs of FIGS. 25 a and 25 b, NaCl (not achaotropic salt) was as effective as guanidine thiocyanate in promotingnucleic acid retention on plain 0.2 micron Anopore membranes, and muchmore effective than guanidine hydrochloride.

3. Example 3

FIG. 26 is a graph of nucleic acid retention vs. pH for plainunmodified, diol modified, and amine (APS) modified 0.2 micron Anoporemembranes. The results were generated in 1 molar NaCl, 50 mM phosphatebuffer, and DNA digest as previously described. As shown in the graph ofFIG. 26, nucleic acid retention was uniformly high and independent ofsurface modification at pH 4, but significant surface modificationeffects were indicated at pH 6 thru 8, with the plain membrane droppingto below 5% nucleic acid retention, and the diol modified membranedropping to about 65% retention. At high pH (10), nucleic acid retentionfell to essentially 0 for the plain and diol modified membranes, but wasstill above 50% for the amine modified membrane.

4. Example 4

The graphs of FIGS. 27-29 show the nature of nucleic acid retention onplain unmodified, amine (APS) modified, and diol modified Anoporemembranes in variable NaCl, with 1 mM Tris buffer, pH 8.0. Each of thesegraph plots % nucleic acid retention or elution vs. NaCl concentrationfor three cases:

Total Membrane Retention (plot is identical to FIG. 24 a);

Rinsed Thru Membrane—after the specified buffer containing nucleic acidwas filtered thru the membrane, an equal volume of identical buffer,without nucleic acid, was filtered thru the membrane and analyzed.

Rinsed From Surface of Membrane—after the membrane was rinsed (RinsedThru Membrane, above), an equal volume of identical buffer, withoutnucleic acid, was placed in the well above the membrane. This buffer wasaspirated and dispensed approximately 10 times onto the membrane torinse localized nucleic acids from the surface. This surface rinse wasanalyzed by absorbance and eluted nucleic acids were calculated.

Plain and diol modified Anopore membranes (FIGS. 27 and 29,respectively) showed an appreciable thru rinse fraction where nucleicacids were apparently retarded rather than retained as they flowed thruthe filter. Little nucleic acid was rinsed from the filter surface afterthis thru rinse. Amine modified Anopore membranes (APS, FIG. 28) showeddifferent behavior. Thru rinse removed little nucleic acid from themodified membrane, while in some cases nearly 60% of the startingnucleic acid was able to be rinsed from the surface of the membrane.

5. Example 5

The graph of FIG. 30 shows the effect of phosphate buffer concentrationon nucleic acid retention or elution for plain unmodified and amine(ethylenediaminopropyltrimethoxysilane, EDAPS) modified membranes.Disodium phosphate was added to 1 M NaCl in the listed concentrations onthe graph and the pH was adjusted to 8.0. Phosphate ions are known tobind to aluminum oxide chromatography packings and may compete for thesame weak binding sites on Anopore membranes as nucleic acids. As shownin the graph of FIG. 30, nucleic acid localization by EDAPS modifiedAnopore membranes was not nearly as sensitive to phosphate concentrationas plain membranes.

Other buffers are also capable of lowering nucleic acid localization onunmodified Anopore membranes. Table 2 shows the percent retention of DNAin 1000 mM NaCl containing the indicated buffers.

TABLE 2 Buffer % Nucleic Acid Localization  1 mM Tris 98.3% 50 mMTris-Borate-EDTA 17.8% 50 mM Phosphate −2.6% 50 mM Bicarbonate  4.6% 50mM Borate  5.7%

6. Example 6

Surface properties are frequently affected by adsorption of species thatblock weak interactions. Such species can be used as membrane coatingmaterials and include surfactants and components of plasma that may bepresent in some applications described herein Table 5 shows the percentlocalization of DNA under conditions that may be found in viral loadassays, including 1 mg/ml Proteinase K enzyme, 2% Tween 20 surfactant,added salt as noted, and with and without 65% EDTA human plasma.

TABLE 3 Membrane Salt Plasma % Nucleic Acid Localization Amine-EDAPSNone 65% 99% Amine-EDAPS None 0% 99% Amine-EDAPS 200 mM NaCl 65% 96%Amine-EDAPS 200 mM NaCl 0% 98% Amine-EDAPS 200 mM GTC 65% 100%Amine-EDAPS 200 mM GTC 0% 96% Unmodified None 65% 42% Unmodified None 0%28% Unmodified 200 mM NaCl 65% 56% Unmodified 200 mM NaCl 0% 97%Unmodified 200 mM GTC 65% 13% Unmodified 200 mM GTC 0% 96%

As indicated in Table 3, nucleic acid localization by EDAPS modifiedmembranes remained consistently high under the varying salt and plasmaconditions, while the unmodified membranes had inconsistent nucleic acidlocalization under the varying conditions.

7. Example 7

In a high sensitivity viral load assay, very small amounts of nucleicacids are quantitized. Table 4 below shows the percent localization ofHIV RNA obtained from pooled clinical samples vs. virion lysis/plasmadigestion reaction conditions. All samples contained a 135 μl patientsample (EDTA plasma), 2 mg/ml proteinase K, 2% Tween 20, and 400 mMguanidine thiocyanate. The samples were incubated for 30 minutes underthe conditions listed in Table 4.

TABLE 4 Incubation Membrane RNA Concentration % Localization Room TempNone 321k copies/ml — Room Temp Plain 274k copies/ml 14.6% Room TempEDAPS  5.7k copies/ml 98.2% 55° C. None 542k copies/ml — 55° C. Plain427k copies/ml 21.2% 55° C. EDAPS  4.1k copies/ml 99.2% RT SonicatedNone 481k copies/ml — RT Sonicated plain 388k copies/ml 19.3% RTSonicated EDAPS  5.8k copies/ml 98.8%

As indicated in Table 4, RNA localization by EDAPS modified membranesremained consistently high under the varying incubation conditions,while the plain unmodified membranes had much lower RNA localizationunder the varying incubation conditions.

8. Example 8

The effect of test sample digestion on human plasma filtration wasinvestigated by use of 300 μl total incubation mixtures containing theingredients and subject to the conditions listed in Table 5 below. Thefilter assembly used included unmodified, 0.2 micron Anopore membranefilters approximately 5 mm in diameter heat fused to the bottom ofmodified Millipore 96 well filter plates, such as shown in theembodiment of FIG. 15 a.

TABLE 5 Proteinase Incubation Incubation Filtration Sample¹ K Enzyme²Surfactant³ Chaotrope Temp.⁴ Time Time⁵ 1 (200 μl None None None RT NonePlugged Plasma) 2 (As Above) 2.3 mg/ml None None 55° C. 20 min. Plugged3 (As Above) 2.3 mg/ml None 0.5 M 55° C. 20 min. Plugged GITC 4 (AsAbove) 2.3 mg/ml 0.5% Triton 0.5 M 55° C. 20 min. 15 sec. GITC 5 (AsAbove) 2.3 mg/ml 0.5% Triton None 55° C. 20 min. 18 sec. 6 (As Above)2.3 mg/ml 2.0% Tween None 55° C. 20 min. 15 sec. 7 (As Above) 1.7 mg/ml1% SDS None 55° C. 30 min. 15 sec. 8 (As Above) 1.0 mg/ml 2% Tween NoneRT 30 min. 14 sec. 9 (As Above) None 2% Tween None RT 30 min. 4+ min.¹Sample - Fresh or frozen EDTA human plasma ²Proteinase K Enzyme -Liquid concentrate (23 mg/ml), Sigma-Aldrich Co. ³Surfactant - MolecularGrade Triton X-100, Tween 20, or SDS (Sodium Dodecyl Sulfate)⁴Incubation Conditions - RT (Room Temperature) 25° C. benchtop; 55° C.water bath. ⁵Filtration Time - The number of seconds to filter 250 μl ofincubate thru the localization membrane filter.

As indicated in Table 5, sample digestion allowed 200 μl plasma samplesto be filtered through 0.2 micron filters in a few seconds in aconvenient multiwell format when a surfactant was used.

9. Example 9

FIG. 31 is a bar graph of a sample digestion/filtration study forcerebral spinal fluid (CSF), human serum, and whole blood. Totalfiltration volume is shown on the left axis, while time to filter thetotal volume through an unmodified 0.2 micron Anopore filter ofapproximate 3.5 millimeter diameter is shown on the right axis. Varioussamples and digestion conditions are shown, including water (control),neat (undigested) CSF, and digested CSF, serum, plasma, and whole blood.Digestion conditions were 15 minutes at 37° C. in a total digestionvolume as shown containing the following:

-   -   CSF, serum, plasma    -   31 mM Tris HCl, 10 mM EDTA    -   800 mM Guanidine Isothiocyanate    -   0.5% Triton X-100    -   5.2% Tween 20    -   0.9 AU Proteinase    -   Whole Blood    -   18 mM Tris HCl, 6 mM EDTA    -   500 mM Guanidine Isothiocyanate    -   0.3% Triton X-100    -   3.0% Tween 20    -   2.0 mg/ml Proteinase K

As is seen, practical volumes of various biological samples arefilterable through small area Anopore filters in reasonable times.

10. Example 10

Genomic nucleic acid extraction and purification from whole bloodincluding PCR amplification was carried out using the followingprocedure. Initially, 10 microliters of EDTA anticoagulated human wholeblood was diluted into 180 microliters of molecular grade watercontaining 10 microliters of a solution of 10% sodium dodecyl sulfate(SDS) for a final concentration of 0.5%. The mixture was briefly mixed,then incubated at room temperature for approximately 15 minutes. Theentire sample was placed into a multiwell filter plate (modifiedMillipore filter plate as previously described) with an integraluntreated 0.2 micron Anopore membrane. The mixture was vacuum filteredover about 30 seconds to dryness then rinsed with about 100 microlitersof distilled water that was again vacuum filtered to dryness through themembrane. The rinsed membrane was essentially white, with very littlebound hemoglobin apparent. Thereafter, 20 microliters of PCR master mix,including primers for the amplification of nucleic acid sequencesinvolved in the genetic coding for human beta globin protein, wasdispensed onto the filter and briefly allowed to resuspend any localizedgenomic DNA that was captured from the initial SDS lysed blood sample.The resuspension was aided by repeatedly dispensing and aspirating the20 microliter master mix onto the membrane. The master mix was aspiratedand transferred to a PCR capillary tube and analyzed by real time PCR(Roche Lightcycler) for nucleic acid sequences involved in the geneticcoding for human beta globin protein. Positive results were obtained.

10 ul whole blood from various de-identified samples was lysed in 200 ultotal volume containing 500 mM Guanidine Isothiocyanate, 0.5% Triton-X100, 20 mM Tris-HCl, pH 6.5, 10 mM EDTA and 0.25 mg/ml Proteinase K(>500 U/mg) for 15 minutes at room temperature (˜25 C). The lysate wassubsequently filtered through an unmodified Anopore membrane (d=˜3.8 mm)by vacuum and then washed with 100 ul of 200 mM NaCl. The filter wasthen dried and the backside passivated with a 1-3 ul bead of DYMAX OP-21flexible plastic bonder which was UV cured for 5 seconds afterapplication. The membrane containing the surface localized DNA derivedfrom the whole blood samples was then added to 50 microliters of PCRmaster mix in a conventional PCR tube containing: 1×PCR buffer (Roche),4 mM MgCl₂ (Roche), 2 mM dATP, dCTP, dGTP and 4 mM dUTP (Perkin Elmer),500 ug/ml BSA (Idaho Technology), 0.5 uM each PCO₃ and PCO₄ primers, 1UHeat Labile Uracil N-Glycosylase (Roche) and 3U FastStart Taq Polymerase(Roche). PCR thermal cycling performed in a conventional thermal cycleras follows: 50 degrees C.×5 min. (UNG), 95 degrees C.×5 min (taqactivation), 40 cycles of [95 degrees C.×3 secs, 55 degrees C.×45 secs,72 degrees C.×60 secs], followed by a final extension at 72 degrees C.for 5 min. 10 ul of each 50 ul PCR reaction was then loaded onto a 2%agarose, TBE gel and electrophoresed. Bands representing β-Globin (˜10bp) or primer dimer (˜35 bp) were visualized after staining gel inethidium bromide and viewing under UV light. Positive results indicatingthe presence of human genomic DNA were obtained. FIG. 32 is a photographof the electrophoretic gel showing amplicons generated by PCRmultiplication of beta globin target regions present on the localizedgenomic DNA contained within the whole blood samples. As is seen, PCRamplification occurred in all cases.

11. Example 11

The ability to use whole blood samples in real time PCR (RT-PCR) forgenetic analysis would simplify laboratory workflow and potentiallyreduce costs and offer other advantages. The whole blood PCRamplification of Example 10 has been expanded to include real timedetection of generated amplicons by performing the analysis in a systemsimilar to that of FIG. 17 b, using the simplified disposable formatshown in FIGS. 18 and 19 as previously described. 10 ul whole blood waslysed then filtered and rinsed through unmodified Anopore filters asgenerally shown in FIG. 18 and as described in Example 10. The driedbottom surface of the Anopore, containing localized genomic DNA derivedfrom the whole blood sample, was sealed by heat welding polypropyleneheat sealing film 1910 directly onto the dry membrane, as shown in FIG.19. 50 microliters of PCR master mix was then added into wells 1901, offormulation as previously described, but included 1:6000 dilution ofSybergreen I dye (Molecular Probes). The tops of wells 1901, containingPCR reaction liquid 1913, was then sealed by clear adhesive tape 1911 orclear caps 1912. This assembly was thermally cycled in a real time PCRinstrument designed to transfer heat directly across the Anopore-sealingfilm surface as depicted in FIG. 17 b. Changes in fluorescence withinthe wells was monitored during thermal cycling by a simplifiedfluorometer composed of an ultra bright blue light emitting diode,photodiode detector, and suitable interference filters, electronics andsoftware. Fluorescence data was evaluated by Lightcycler (Roche)software. Thermal cycling was accomplished by employing a Perkin Elmer480 instrument modified to allow remote operation of a custom built flatthermal cycling area. Thermal cycling profile was similar to Example 10(99 degrees C.×20 sec., 50 degrees C.×3 secs., 75 degrees C.×60 seqs.).Melting analysis was done by ramping the temperature from 65 degrees C.to 99 degrees C. over a one hour period.

A real time amplification curve for Example 11 is shown in FIG. 33,while a melting curve is shown in FIG. 34. Similar results are shown fora lightcycler in FIG. 34. The figures show unequivocal evidence ofspecific PCR amplification and identification of the beta globin genefrom membrane localized DNA from digested whole blood in a simplifiedreal time format.

12. Example 12

Human genomic DNA can be directly visualized by fluorescent staining. 10ul of purified human white blood cells, concentration greater than 1million cells/ml, was added to 190 ul of TE buffer containing 0.5%sodium dodecyl sulfate and incubated for 20 minutes at 56 degrees C. tolyse the white blood cells and release DNA. This solution was diluted toa final concentration, based on the starting 10 ul of cell solution, of1 to 400,000 in a solution composed of 1 to 10,000 dilution of SyberGold in TE buffer. The dilute DNA solution was incubated in the SyberGold solution for a few minutes, then 50 ul filtered across aNickel-Boron modified Anopore membrane according to Example 22. Themembrane, containing localized human genomic DNA, was briefly rinsedwith TE buffer then visualized on an instrument similar to shown in FIG.9 and further described in Example 13. A typical image of human genomicDNA according to this example is shown as FIG. 11.

13. Example 13

Nucleic acids were optically analyzed. Purified calf thymus DNA ofapproximately 13,000 base pairs was serial diluted at the relativeconcentrations of 1 to 128 into filtered 10 mMolar Tris-HCl, 1 mMolarEDTA, pH 7.5 buffer containing 100 nanomolar YOYO-1 nucleic acid dye.After 5 minutes of room temperature incubation, 100 microliters of thedyed nucleic acid samples were filtered through an unmodified 0.2 micronAnopore membrane of approximately 3 mm active filter diameter that hadbeen heat fused onto a black polypropylene holder. The filter was imagedwith a fluorescence microscope of the configuration shown in FIG. 9,using a 20 milliwatt air cooled argon laser operating single line at 488nm, and a 1024×1058 pixel backthinned CCD camera cooled to −10° C. witha 0.3 NA 10× flat field microscope objective. The DNA is easilyvisualized with approximately 5 second exposure. The images were signalprocessed to sharpen edges, remove obvious non nucleic acid defects,corrected for background fluorescence, etc., then the remainingprocessed DNA spots were numerically counted. The processed images areshown in FIG. 12, the resulting simple dilution curve in FIG. 13, andthe dilution curve corrected for instrument and system parameters inFIG. 14.

14. Example 14

Theoretical modeling was carried out to evaluate various detectionparameters for a confocal scanning detection system such as depicted inFIG. 8. The following assumptions were made for the theoreticalmodeling.

1. Nucleic acids contained in a 1 ml sample can be bound to a 1 cm²area.

2. A false positive rate due to counting error of 1 per cm² isacceptable.

3. Nucleic acids are stained with 25 or 100 dye molecules/nucleic acid.

4. Each fluorescent dye molecule generates a detected signal of 50,000photons/second when in the detection zone. This is achieved atillumination densities of 100 kW/cm².

5. Noise is from background sources such as instrumentation noise, Ramanscattering, plastic fluorescence, etc. Additionally, surface nonspecificbinding (NSB) from nucleic acid staining results in uniformlydistributed, isolated (nonclumped) dye molecules with a very lowprobability of multiple dyes/scanning area. Inadvertent staining ofsurface bound nonspecific nucleic acids, etc. that gives a clumpedfluorescent signal has not been considered.

The theoretical modeling was based on the following detectionconditions. A fluorescently labeled nucleic acid is bound to an opticalsurface. A confocal scanning epifluorometer intensely illuminates thebound nucleic acid causing it to fluoresce. Emitted fluorescent photonsfrom the nucleic acid are captured and counted. Additionally, theepifluorometer detects a background count rate from scattered light,instrument noise, NSB, etc. The specific signal from the fluorescentnucleic acid is present as a small burst of photons occurring in aninterval defined by the scanning speed and spot size. As a typicalexample, bursts of 3 to 20 photons occurring in 0.5 to 10 microsecondswere calculated. The background or noise signal follows a Poissondistribution and has a finite probability of occurring in bursts thatare indistinguishable from the specific nucleic acid signal.

Required detection parameters (SNR, scan speed, spot size, etc.) weredetermined by setting the fluorescent nucleic acid signal equal to themaximum statistical noise burst count occurring during the 1 cm² scan.This results in a statistical false positive count of 1/cm² frombackground noise. That is to say, a given scan speed and scan spot sizeresults in a photon burst time, which is the time it takes for theillumination spot to sweep over an immobilized fluorescent nucleic acid(e.g., about 0.5 to about 10 microseconds). The number of photonscounted by the detector per photon burst time is used to discriminatebetween nucleic acids and background. Background noise contains smallbursts due to statistical fluctuations. The minimum required nucleicacid signal (photons/burst time) is set equal to the maximum noisesignal (photons/burst time) that may statistically occur once during thetotal scan time. Accordingly, the instrument counts 1 nucleic acid eachtime this specified burst rate (for instance 5 photons in 2microseconds) is exceeded. Statistically, noise should account for 1such occurrence per 1 cm² scanned, or 1 false positive per 1 ml sample.

The following equations and mathematical relationships were utilized inthe theoretical modeling that was carried out in the examples.

1. Poisson Random Variable Equation:

P _((I)) =e ^(−G) G ^(I) /I!

-   -   where I is the number of photons in a given interval;        P is the probability of I photons occurring in a given interval;        and        G is the average probability of photons occurring in a given        interval.

2. General Relationships:

-   -   T=seconds/1 cm² scan.    -   D=spot dimension, microns.

Photon Burst Time (microseconds)=T(D²)/1×10⁸

where 1×10⁸ is the number of microns² per cm².

Scan Speed (cm/sec)=1×10⁴/TD

Nucleic Acid Signal (photon counts/photon burst time)=FRTD²/1×10⁸

where F is the number of fluorescent dye molecules/nucleic acid;

and R is the detected photon count rate/dye molecule (50,000photons/second/molecule assumed).

Background Signal (photon counts/photon burst time)=BTD⁴/1×10⁸

where B is the average background signal in photoncounts/second/micron².

NSB Signal (photon counts/photon burst time)=ZTRD⁴/1×10⁸

where Z is the number of fluorescent dye molecules/micron²non-specifically bound to the optical surface.

With the signals expressed on a photon burst time basis, they are usefulin the Poisson Random Variable equation as follows:

I=Nucleic Acid Signal;

this is the minimum nucleic acid signal (photons/burst time) for 1 falsepositive/cm².

-   -   G=Background Signal+NSB Signal;

this is the average photon count/burst time for all non-nucleic acidsources.

P<D ²/1×10⁸

This condition sets the statistical occurrence of false positives toless than 1 in a complete 1 cm² scan. With these relationships, curvesof nucleic acid counting performance under various conditions weregenerated as described in the following examples.

15. Example 15

FIG. 35 is a graph of scan time vs. spot dimension for the followingfive detection conditions:

1. 100 dye molecules/nucleic acid, 10 khz/micron² noise, 0 dyemolecules/micron NSB;

2. 1100 dye molecules/nucleic acid, 40 khz/micron noise, 0 dyemolecules/micron² NSB;

3. 1100 dye molecules/nucleic acid, 40 khz/micron noise, 1 dyemolecules/micron NSB;

4. 25 dye molecules/nucleic acid, 10 khz/micron² noise, 0 dyemolecules/micron NSB; and

5. 25 dye molecules/nucleic acid, 40 khz/micron² noise, 1 dyemolecule/micron² NSB.

As shown in the graph of FIG. 35, by setting a scan time limit of 200seconds, manifestation of all but the noisiest 25 dye molecules/nucleicacid case is possible with a 2 micron spot. No data is presented forthis case with spot dimensions larger than 2 microns, since the curveturns rapidly upward for spot dimensions between 2 and 3 microns as theSNR approaches 2.

16. Example 16

FIGS. 36-39 are graphs of scan time vs. signal/noise, where:

signal to noise ratio (SNR)=Nucleic Acid Signal/Background Signal+NSBSignal

The graph of FIG. 36 was generated for the conditions of 100 dyemolecules/nucleic acid at various spot dimensions, and between 2 and 500SNR. The graph of FIG. 37 was generated for the conditions of 100 dyemolecules/nucleic acid at various spot dimensions, with an expandedscale between 2 and 100 SNR. The graph of FIG. 38 was generated for theconditions of 25 dye molecules/nucleic acid at various spot dimensions,and between 2 and 500 SNR. The graph of FIG. 39 was generated for theconditions of 25 dye molecules/nucleic acid at various spot dimensionswith an expanded scale between 2 and 100 SNR.

The actual values of SNR at the left end of each of the graphs of FIGS.36-39 are 2.5, 3.0, 4.0, 5.0, and 10.0. The graphs of FIGS. 36-39 revealthat scan times increase dramatically at SNR lower than about 10. Scantimes for SNRs lower than 2.5 have not been calculated other than toobserve as SNR ratio falls lower than 2.5, scan times become enormous.

Based on the theoretical modeling, fastest scanning is achieved with thelargest spot until SNR issues begin to dominate. Referring again to FIG.37, the point shown for 25 dye molecules/nucleic acid, 40 kHz/micron2noise, 1 dye molecule/micron2 NSB at 2 micron spot dimension, has an SNRof 3.472. This is the same SNR found at 4 micron spot dimension for the100 dye molecule/nucleic acid, 40 kHz/micron2 noise, and 1 dyemolecule/micron2 NSB case. Both curves are relatively flat. A completeplot of theses curves would show a strong upturn in scanning time,approaching infinity, as the SNR falls towards 1. It appears that theminimum useful SNR is approximately 3.

As long as NSB is present as isolated dye molecules, it is additive withgeneral background noise and the SNR prediction is valid. Any mechanismthat causes dye molecules to concentrate into a region similar in sizeto a spot size used for scanning, such as staining of surface boundnonspecific nucleic acids, may result in the erroneous counting of anonspecific nucleic acid as a specific nucleic acid.

17. Example 17

FIG. 40 is a high magnification negative image photomicrograph of lambdaphage DNA of 48,502 base pair length localized on an unmodified 0.2micron pore size Anopore membrane filter and stained with YOYO-1 dye,which was imaged according to the technique described in Example 3. FIG.40 clearly shows the DNA on the surface of the membrane filter, with DNAmolecular shape readily apparent. The lighter elongated shapes in FIG.40 correspond to fully extended, linearized DNA and the darker globularshapes correspond to collapsed molecular conformations. Additionally,intermediate structures are readily apparent, where a linear tail isseen in conjunction with a dark spot, representing partial molecularelongation.

18. Example 18

The viral load of a sample is to be determined by nucleic acid counting.A numerical example of correlating the initial sample viral loadconcentration from the counted nucleic acids may be based on thefollowing values:

1. Virion Lysing Efficiency 99% 2. Sample volume 200 ul 3. Nucleic AcidCapture Efficiency 85% 4. Nucleic Acid Labeling Efficiency 95% 5.Nucleic Acid Detection Efficiency 99% 6. Detected Membrane Percentage25% 7. System Linearity 95%

During the assay, an image of the analysis membrane can show 1,000detected discrete labeled nucleic acids. Starting with parameter 7 andworking backwards, 1000 detected discrete nucleic acids equates to 1,053(1000/.95) actual nucleic acids due to system linearity. That is to say,the detector cannot accurately count all nucleic acids at certainconcentrations. Since only 25% of the entire membrane filtration areawas imaged, the 1,053 nucleic acids counted represents 4,212(1,053/0.25) nucleic acids on the entire filter from the sample.Additionally, only a percentage of the nucleic acids can be detected forother reasons, and only a percentage of the nucleic acids were labeledto allow for detection. Accordingly, the 4,212 nucleic acids represent4,478 (4,212/0.99×0.95) nucleic acids that were on the filter. The 4,478nucleic acids on the filter actually represent 5,269 (4,478/0.85)nucleic acids that actually flowed through the filter, because only 85%of those nucleic acids were actually retained on the filter. The 5,269nucleic acids that flowed through the filter were derived from 200 ul ofinitial sample, or a concentration of 10,538 (5,269/0.2 mls) nucleicacids/ml of sample. Finally, lysis conditions are known to producesuitable nucleic acids from target virions at 95% efficiency.Accordingly, the 10,538 nucleic acids/ml calculated represents 10,644(10,538/0.99) actual virions present in a milliliter of patient sample.

19. Example 19

0.2 micron 47 millimeter diameter Anopore membranes were dye modified asfollows: 3 milliliters of EDAPS (ethylenediaminopropyl trimethoxysilane)was dissolved into 27 milliliters of high purity water with mixing. Thesolution was filtered through a 25-millimeter diameter Anotop filter(Whatman, 0.2 micron) to remove all particulate, gelled reagents,precipitate, etc. Dry Anopore membranes were then completely immersed inthe filtered solution. The solution, including the immersed membranes,was sonicated briefly, then placed in a vacuum oven and the airevacuated to aid in membrane wetting. After 5 minutes total immersiontime, the wet membranes were carefully removed from the solution andrinsed twice by immersion into 100 milliliters of high purity water withgentle mixing to remove unbound silane reagent. The membranes weresimilarly rinsed in 100 milliliters of 5×TE buffer to neutralize surfacebound amino groups, then again in 100 milliliter of high purity water.

The moist membranes were dehydrated for approximately 3 hours in a warm(100 degree C.) oven under vacuum. The dry EDAPS membranes were thenimmersed in a freshly prepared and filtered solution of amino reactivedye (Procion MX, Reactive Blue 4, DTAF, etc.) containing approximately0.5 gram of dye in 50 milliliters of high purity water. Air bubbles wereagain removed by sonication and vacuum. The membranes remain in the dyesolution at room temperature for approximately 4 hours. The darklycolored membranes were rinsed repeatedly with high purity water thendried. The stained, dry membranes are hydrophilic and easily filterwater.

20. Example 20

0.2 micron 47 millimeter diameter Anopore membranes were modified byinclusion of carbon black optical pigments as follows:

Approximately 0.2 grams of carbon black (Raven 5000 Ultra 2, ColumbianChemicals, Inc.) were mechanically dispersed into 1 milliliter of highpurity water containing approximately 2% Tween 20 surfactant. Theresulting black pigment dispersion was applied to the membrane surface,allowed to sit for a few minutes, then rinsed from the membrane withwater. The resulting black membrane is optically opaque and somewhathydrophobic but able to filter water.

21. Example 21

Anopore membranes were optically modified by electroless metaldeposition as follows: Metal alloys suitable for electroless depositionare known to those skilled in the art and include nickel, phosphorous,boron, gold, palladium, silver, etc. Metallization must be controlled toallow optical modification to take place while retaining fluidfiltration properties. The metallization process on Anopore membrane isgenerally a two-part process:

Activation-Anopore is generally non-reactive to metallization solutions.Accordingly, the membrane was activated by incorporation of palladiumprior to metallization. This was accomplished as follows:

Palladium chloride was dissolved in dimethylsulfoxide (DMSO) at a finalconcentration of 5-milligrams/milliliter by stirring and gentle heating.0.5 milliliters palladium chloride/DMSO solution was dissolved into 49.5milliliters of acetone (1% solution). Dry Anopore membranes were fullyimmersed in the palladium chloride/DMSO/acetone solution for 1-2 minutesthen removed. Hanging droplets were wicked from the membrane surface,then the membrane was allowed to air dry completely. Immersing theacetone moist membrane into diethyl ether to quickly remove theacetone/DMSO and precipitate the palladium chloride can also beperformed. Additionally, water may be substituted for the acetone asdiluent. In this case, acetone is used in place of diethyl ether toquickly dehydrate the membrane and precipitate the palladium chloride.Palladium chloride, present within the membrane as a uniform dispersionof particles, must be chemically reduced to metallic palladium byreaction with known reducing reagents, such as sodium borohydride,dimethylaminoborane, sodium hypophosphite, etc. prior to electrolyticmetallization. In many cases this is accomplished in the finalmetallization reaction, where the palladium salt is reduced to catalyticmetal by reaction with the metallization bath-reducing reagent prior tobulk metal deposition.

Metallization-Many formulations exist for electroless metallization. Byway of example, electroless nickel alloys, containing either phosphorousor boron as alloying agents, are widely known. Both initially formoptically black deposits (“black nickel”) and are useful in theembodiments described herein.

Black nickel/phosphorous pigmentary deposits were formed by immersingpalladium chloride activated Anopore membranes in a solution containing30 grams/liter nickel chloride, 10 grams/liter sodium hypophosphite, 50grams/liter ammonium chloride, pH 8-10 at a bath temperature of 90degrees C. Upon immersion, the Anopore membrane slowly turns brown thenblack. Within approximately 5 minutes, the membrane is totally blackwith an optical absorption of greater than 3.0. These blacknickel/phosphorous membranes appear slightly less hydrophilic thanunmodified Anopore, but retain very high water flow rates forfiltration.

Black nickel/boron pigmentary deposits were formed by immersingpalladium chloride activated Anopore membranes in a solution containing25 grams/liter nickel sulfate, 15 grams/liter sodium acetate, 4grams/liter dimethylaminoborane, pH 5.9 at room temperature. Thisreaction proceeds essentially as described for the nickel/phosphoroussystem. In both cases, the membranes initially turn black, buteventually (10+ minutes) become metallic and non-porous as the nickelalloy continues to deposit within the pores and on exterior surfaces.FIG. 42 is an electron micrograph image of the interior of Anoporemembrane modified according to Example 21 to contain black nickel/borondeposits. As is seen, the metallic deposits are exceptionally small,isolated particles and do not appear to materially effect membrane flowproperties.

FIG. 41 shows optical absorption versus wavelength for three organic dyemodified Anopore membranes and one Anopore membrane withnickel-phosphorous deposited on the membrane (the deposited pigmentmembrane) according to Examples 19 and 21.

FIG. 42 is an electron micrograph of the interior of an improved Anoporemembrane prepared by the method described in Example 21. Bright spotsare pigmentary inclusions of nickel-boron alloy deposited according toExample 21. Table 6 shows membrane autofluorescence results for a plainAnopore membrane, the three organic dye modified membranes, and thedeposited pigment membrane. Data was taken with a custom designed darkfield epifluorometer as shown in FIG. 14. The organic dyed membraneswere modified according to Example 19 with Procion MX Jet Black,Reactive Blue 4, or Procion MX Red and the deposited pigment membraneaccording to Example 21 by inclusion of black nickel/phosphorous. Yellowfluorescent polystyrene beads (Molecular Probes, Inc.) having a diameterof 0.2 microns diameter were imaged against the membranes and true beadfluorescence was determined. The background signals were also measured.The data demonstrates substantial reduction in autofluorescence ofAnopore membranes with non-fluorescent organic dye or deposited pigment.

TABLE 6 Corrected Membrane 0.2 Micron Beads Membrane Bead/MembraneModification Yellow Fluorescent Signal Signal Ratio Unmodified 660,00081,250 8.123 Membrane Black Pigment 320,000 500 640 Membrane OrganicBlack 270,000 20,500 13.17 Membrane Organic Blue 290,000 10,500 27.62Membrane Organic Red 280,000 18,000 15.55 Membrane

Table 7 shows NSB detectability reduction results for plain as well asthe black pigmented (nickel/phosphorous) Anopore membranes previouslydescribed. Two examples are shown. Line 1 shows data for fluorescentprotein NSB detectability reduction. A fluorescent protein solution wasprepared containing the following:

100 nanomolar R-Phycoerythrin (Molecular Probes, Inc.)1×TE buffer (10 millimolar Tris, 1 millimolar EDTA, pH 8.0)

1% BSA

40 microliters of the above described R-phycoerythrin solution wasfiltered through 150 inch diameter flow area modified and unmodified 0.2micron Anopore membranes then rinsed once with 40 microliters of 1%BSA-1×TE buffer then rinsed with 200 microliters of plain 1×TE buffer.Fluorescent measurements were made on the instrument of FIG. 9 with theemission filter changed to a 595-nanometer center wavelength by 50nanometer bandpass.

Line 2 shows data for fluorescent bead NSB detectability reduction. Thiswas identical in procedure and reagents to Line 1 with 0.02 micronyellow fluorescent microbead solution (Molecular probes, Inc., 748dilution of 1% solids stock solution, 10 nanomolar equivalent beadconcentration in 1% BSA 1×TE buffer) replacing the fluorescentR-phycoerythrin solution. Fluorescence measurements were made on theinstrument of FIG. 9. Both examples demonstrate substantial reduction indetected fluorescence from non-specifically bound substances.

TABLE 7 Black Pigment NSB Detectability NSB Unmodified 0.2 Modified .2Ratio, unmodified/ Species Micron Anopore Micron Anopore modifiedProtein 6,400 48 133 .02 Micron 255,000 858 297 Beads *Fluorescent datacorrected for membrane autofluorescence.

Pigment modified membranes may be additionally modified by silanizationreactions as previously described for plain membranes to impart usefulsurface characteristics such as NSB reduction, molecular capture,covalent attachment, etc.

22. Example 22

Nucleic acid detection and quantification by enzyme linked fluorescence(ELF) may be performed as follows:

20 complimentary biotinylated oligonucleotide probes were customsynthesized to lambda phage DNA. These probes were designed to bindspecifically to the target DNA as two groups of 10 probes each. Theprobes in each group were designed to bind with a typical spacing ofapproximately 200 base pairs. This resulted in two regions of high probebinding density on the lambda DNA target. Calculations showed if thelambda DNA was fully linear, the 10 probes in each region would becontained in a length of approximately 0.6 microns. The two regions wereseparated by approximately 25000 base pairs, or 7-8 microns for the fulllinear case.

Lambda DNA was prehybridized to the above described probes as follows:

One ul of purified lambda phage DNA (500 ug/ml) was added to 499 ul ofPCR Fast Start Buffer (Roche) containing 4 mM MgCl₂ and 200 nM of eachof the 20 above described probes. This mixture was heated to 95 DegreesC. for 10 minutes, cooled to 56 degrees C. for 5 minutes, cooled to 37degrees C. for 2 minutes, then cooled to room temperature. Unreactedprobes were removed with a Qiagen PCR clean up kit followingmanufacturer's directions. The final lambda DNA, containing 20hybridized biotinylated probes, was diluted to 1 ug/ml in TE buffer andfrozen at −20 degrees C.

The labeled lambda DNA was visualized as follows:

Nickel-Boron modified Anopore membrane was prepared according to Example21 and configured as shown in FIG. 18 a into an assembly with 12individual wells. This assembly was placed into a simple vacuum housingthat permitted vacuum filtration by sealing tightly against the Anoporemembrane. 20 ul of a 1:200 dilution of the above described prehybridizedlambda DNA in TE buffer was filtered in several wells, while 20 ul ofplain TE buffer was filtered in different wells. 10 ul of TBT buffer (10mM Tris, 100 mM NaCl, 1% BSA, 0.1% Tween-20, pH 7.8) was filteredthrough all wells, then 10 ul additional TBT buffer was added andincubated in all wells for 5 minutes to block effects of non specificprotein binding on the modified Anopore membrane. The TBT incubate wasfiltered, then the filter assembly was removed from the vacuum housingthen reinstalled with Parafilm covering the Anopore membrane. TheParafilm seals the Anopore filter to prevent liquid flow duringsubsequent steps. 10 ul of a 1:50 dilution of streptavidin-alkalinephosphatase (SAP, Molecular Probes ELF kit for in situ hybridization) inTBT buffer was added to each well and incubated at room temperature for15 minutes. The wells were aspirated to dryness, 10 ul of TBT bufferadded to each well then again aspirated to dryness, then 10 ul of washbuffer (Molecular Probes ELF kit) then again aspirated to dryness. Thefilter assembly was removed from the vacuum housing and the parafilmremoved. The assembly was then reinstalled in the vacuum housing. 100 ulof wash buffer was filtered through each well to dryness. 10 ul of ELFsubstrate, prepared according to the directions supplied with theMolecular Probes kit, was added to each well and incubated for apredetermined time. Briefly, the ELF substrate is a 1:10 dilution of asubstrate reagent in development buffer that is filtered. Two additionalreagents are added after filtration, each at 1:1,000 dilution. Each ofthese reagents and buffers is proprietary to Molecular Probes and arecontained in the ELF kit.

After the timed ELF development incubation, each well is rinsed andaspirated with 10 ul wash buffer followed by 10 ul of distilled water.The wells are visualized on a detector as shown in FIG. 9 with theexcitation source a mercury compact arc lamp with suitable filters toallow illumination at nominally 365 nm and detection filters set at 527nm.

The results of this example are shown in FIG. 6, clearly showing adifference in the developed ELF signal between the wells containinglocalized DNA and the wells containing no DNA. Additionally, a timeeffect of ELF incubation is clearly visible as previously described.

23. Example 23

Anopore membranes were optically modified by an aqueous basedelectroless metallization process as follows:

All reagents ACS grade or better. The following stock solutions wereprepared. Tin chloride/dimethyl sulfoxide was prepared by dissolving1.00 grams SnCl₂ in 10.0 milliliters DMSO at room temperature for afinal stock concentration of 100.0 milligrams/milliliter. Palladiumchloride/dimethyl sulfoxide was prepared by dissolving 50 milligrams ofPdCl₂ in 10.0 milliliters DMSO at room temperature for a final stockconcentration of 5.0 milligrams/milliliter.

The following working baths were prepared. Working Bath 1—tinpreactivation bath: 1.0 milliliter of tin chloride/dimethyl sulfoxidestock solution was dissolved into 99.0 milliliters of water for a finaltin chloride concentration of 1.0 milligram/milliliter. This solutionwas stable for several days, but may eventually precipitate. WorkingBath 2—palladium activating bath: 2.0 milliliters of the palladiumchloride/dimethyl sulfoxide stock solution was dissolved into 98.0milliliters of water for a final palladium chloride concentration of 0.1milligrams/milliliter. This solution was stable for several days, butmay eventually precipitate. Working Bath 3—Electroless nickel bath: 25.0grams nickel sulfate (NiSO₄.6H₂O), 15.0 grams sodium acetate(NaC₂H₃O₂.3H₂O), 4.0 grams dimethylamine borane (C₂H₁₀NB), and 2.0milligrams lead acetate (Pb(C₂H₃O₂)₂-3H₂O) were dissolved into 1.0 literwater. The pH was adjusted to 5.9 with acetic or sulfuric acid.

Procedure:

1. A 47 millimeter diameter 200 nanometer pore size Anopore™ filter discwas immersed into Working Bath 1 for 1-2 minutes. 80 milliliters ofsolution in a 100 milliliter glass beaker was used to keep the filterdisc nearly vertical during the procedure without solution stirring.This step does not seem to be very sensitive to filter disc orientation,solution agitation, or residence time in the bath. Dry filter discsdirectly from the box without any pretreatment can be used. It is alsopossible to use wet filter discs from a previous processing step.2. Remove the filter disc from Working Bath 1 and immerse it into waterfor approximately 1 minute to remove unreacted reagents. 500 millilitersof deionized water was used and the disc was gently and repeatedlyimmersed into the water. This rinse step removes non-adsorbed tin ionsthat may cause unwanted reactions with subsequent processing steps.3. Remove the filter disc from the water rinse and immerse in WorkingBath 2 for 1-2 minutes. This step is performed in a similar or identicalmanner as that in step 1. Not wishing to be bound by theory, it isbelieved that palladium ions are reduced to surface catalytic palladiummetal atomic clusters by reaction with adsorbed tin(II) ions.4. Remove the filter disc from Working Bath 2 and immerse in water aspreviously described in step 2 to remove unreacted reagents. This rinsestep removes non adsorbed palladium that may cause plating bathdecomposition.5. Remove the filter disc from the water rinse followed by immersing thedisc in Working Bath 3 for approximately 5 minutes with mild agitationuntil black. We generally continue this reaction until the filter discis sufficiently black so as to totally obscure the light from a halogenlight bulb when viewed through the black filter disc. As arepresentative procedure, 80 milliliters of solution in a 100 milliliterbeaker with a small stir bar for agitation is used. The filter disc isgenerally vertical and turns dark uniformly, with slight surfacevariation. Without stirring, hydrogen bubbles released from the filtersurface create vertical dark “tracks” due to local mixing. Fifteen blackdiscs have been prepared from 80 milliliters of Working Bath 3 solutionwithout adding additional reagents. In a production setting, thecomposition of Working Bath 3 would be continuously monitored andreagents replenished as required. With adequate water rinsing betweenworking baths, no spontaneous decomposition of Working Bath 3 wasobserved.6. Remove the filter disc from Working Bath 3 and immerse it in water aspreviously described to remove unreacted reagents. As the final rinse,this rinsing step can be repeated as much as necessary to ensure thatall working solution reagents have been removed.7. Remove the filter disc from the final rinse and dry. Filter discs aredried either in a vacuum chamber under reduced pressure, on the bench atroom temperature, or with heated circulating air.

24. Example 24

Efficiency of PCR based detection of nucleic acids localized tounmodified Anopore membrane, with and without destabilization, wasperformed by one of the following 3 methods:

Method 1: 5 nanograms of purified human genomic DNA was dissolved in 100microliters of water containing 100 millimolar NaCl was filtered through2.5 millimeter diameter unmodified AOM contained in a filter assembly asshown in FIG. 16, followed by filtering 100 microliter water to rinsethe AOM of unlocalized nucleic acids. The outlet of the filter assemblywas capped then 10 microliters of destabilization buffer was added andthe top capped. The sealed filter assembly was then either sonicated forapproximately 1 minute in a laboratory water bath sonicator or heated to25-100° C. for approximately 1 minute in a thermal cycling instrument.The top cap was removed, and 40 microliters of a PCR master mixcomprising 1× iQ Supermix (Bio-Rad, Inc), 0.5 micromolar each PCO₃ andPCO₄ primers for human betaglobin, 1/30000 final dilution of Syber Green1 dye (Molecular Probes, Inc), and 0.5 milligram/milliliter BSA wasadded and the top recapped. The filter assembly was placed in an iCycler(Bio-Rad, Inc) real time PCR instrument and cycled as follows:

Amplification

95° C. for 3:00 minutes

45 cycles of the following:

95° C. for 20 seconds Denaturation

55° C. for 30 seconds Annealing

72° C. for 30 seconds Elongation

80° C. for 10 seconds Detection

Melt Analysis

95° C. for 30 seconds

70° C. for 10 seconds

Ramp temperature in 45 steps at 0.5° C./step, hold for 10-seconddetection

Crossing thresholds are computed by the instrument and are used tocalculate PCR amplifiable localized nucleic acid.

Method 2: 5 nanograms of purified human genomic DNA was dissolved in 100microliters of water containing 100 millimolar NaCl was filtered through2.5 millimeter diameter unmodified AOM contained in a filter assembly asshown in FIG. 16, followed by filtering 100 microliter water to rinsethe AOM of unlocalized nucleic acids. The outlet of the filter assemblywas capped and 40 microliters of a PCR master mix comprising 1× iQSupermix (Bio-Rad, Inc), 0.5 micromolar each PCO₃ and PCO₄ primers forhuman betaglobin, 1/30000 final dilution of Syber Green 1 dye (MolecularProbes, Inc), 0-10 millimolar destabilization ions (phosphate, borate,etc) and 0.5 milligram/milliliter BSA was added and the top capped. Thefilter assembly was placed in an iCycler instrument and amplified asdiscussed above in Method 1.Method 3: 5 nanograms of purified human genomic DNA was dissolved in 100microliters of water containing 100 millimolar NaCl and was filteredthrough 4.0 millimeter diameter unmodified AOM contained in a filterplate as shown in FIG. 18, followed by filtering 100 microliter water torinse the AOM of unlocalized nucleic acids. The 4 millimeter AOMcontaining the localized nucleic acids was “punched” or broken from theplate into a conventional 0.5 milliliter thin wall PCR tube, 10microliters of destabilization buffer was added and the top capped. ThePCR tube was then either sonicated for approximately 1 minute in alaboratory water bath sonicator or heated to 25-100° C. forapproximately 1 minute in a thermal cycling instrument. The top cap wasremoved, and 40 microliters of a PCR master mix comprising 1× iQSupermix (Bio-Rad, Inc), 0.5 micromolar each PCO₃ and PCO₄ primers forhuman betaglobin, 1/30000 final dilution of Syber Green 1 dye (MolecularProbes, Inc), and 0.5 milligram/milliliter BSA was added and the toprecapped. The PCR tube was placed in an iCycler (Bio-Rad, Inc) real timePCR instrument and amplified as discussed under Method 1.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the compounds, compositions and methods described herein.

Various modifications and variations can be made to the microporousmaterials, methods, and articles described herein. Other aspects of themicroporous materials, methods, and articles described herein will beapparent from consideration of the specification and practice of themicroporous materials, methods, and articles disclosed herein. It isintended that the specification and examples be considered as exemplary.

1-29. (canceled)
 30. A method for detecting an analyte, comprising (a)passing a fluid sample comprising the analyte through or into amicroporous material, wherein the analyte is localized near the surfaceof the microporous material; (b) destabilizing at least some of thelocalized analyte; and (c) detecting the destabilized analyte.
 31. Themethod of claim 30, wherein the destabilizing step comprises (1)contacting the localized analyte with a base; (2) agitating thelocalized analyte; (3) heating the localized analyte; (4) contacting thelocalized analyte with one or more ionic species; (5) contacting thelocalized analyte with one or more enzymes; (6) applying an electricalcharge or ionizing energy to the localized analyte; (7) contacting thelocalized analyte with an organic solvent, or a combination of any ofthe steps above.
 32. The method of claim 30, wherein the destabilizingstep comprises contacting the localized analyte with a base.
 33. Themethod of claim 32, wherein the base has a pH of greater than
 9. 34. Themethod of claim 32, wherein the base has a pH of from 10 to
 12. 35. Themethod of claim 32, wherein the base comprises an inorganic base or abuffer.
 36. The method of claim 30, wherein the destabilizing stepcomprises agitating the localized analyte.
 37. The method of claim 36,wherein the agitation step comprises sonication, vortex mixing, orpumping.
 38. The method of claim 30, wherein the destabilizing stepcomprises heating the localized analyte above 25° C. from 60° C. to 100°C.
 39. The method of claim 30, wherein the delocalizing step comprisescontacting the localized analyte with one or more ionic species.
 40. Themethod of claim 39, wherein the ionic species comprises phosphate ions,borate ions, or a combination thereof.
 41. The method of claim 30,wherein the destabilizing step comprises contacting the localizedanalyte with one or more enzymes.
 42. The method of claim 41, whereinthe enzyme comprises a restriction endonuclease, a nick enzyme, or ahelicase.
 43. The method of claim 30, wherein the microporous materialcomprises an inorganic electrochemically formed membrane or an etchmembrane.
 44. The method of claim 30, wherein the microporous materialcomprises micropores having a diameter of less than 0.2 microns.
 45. Themethod of claim 30, wherein the microporous material comprisesmicropores having a diameter of from about 0.02 microns to about 0.2microns.
 46. The method of claim 30, wherein the microporous materialcomprises aluminum or titanium that has been anodized.
 47. The method ofclaim 30, wherein the microporous material comprises a metal oxide. 48.The method of claim 47, wherein the metal oxide comprises aluminumoxide, zirconium oxide, titanium oxide, zeolite, or a combinationthereof.
 49. The method of claim 30, wherein the microporous materialcomprises the pigmented composite prepared by a method comprising: (a)contacting a microporous material with a tin compound to produce a firstcomposite, and (b) contacting the first composite with a pigmentcomprising an elemental metal, a metal oxide, a metal alloy, a metalsalt, or a combination thereof.
 50. The method of claim 30, wherein themicroporous material comprises the pigmented composite of comprising amicroporous material, a tin compound, and at least one pigment, whereinthe tin compound and pigment are incorporated in the microporousmaterial.
 51. The method of claim 30, wherein the microporous materialcomprises a composite comprising a microporous material and a pigment,wherein the pigment is incorporated in the microporous material.
 52. Themethod of claim 51, wherein the pigment is covalently attached to themicroporous material.
 53. The method of claim 52, wherein the pigment isattached to the microporous material by a linker.
 54. The method ofclaim 53, wherein the linker comprises an organosilyl group.
 55. Themethod of claim 52, wherein the pigment is attached to the microporousmaterial by a hydroxyl group, a carboxyl group, a sulfhydryl group, anamine group, or a combination thereof present on the surface of themicroporous material.
 56. The method of claim 52, wherein the pigmentcomprises an organic dye.
 57. The method of claim 56, wherein theorganic dye comprises an isothiocyanate, a triazine, or an ester. 58.The method of claim 51, wherein the microporous material comprises aceramic, a metal, carbon, or glass.
 59. The method of claim 51, whereinthe microporous material comprises a metal oxide.
 60. The method ofclaim 59, wherein the metal oxide comprises aluminum oxide, zirconiumoxide, titanium oxide, a zeolite, or a combination thereof.
 61. Themethod of claim 51, wherein the microporous material comprises aninorganic electrochemically formed material or an etch material.
 62. Themethod of claim 51, wherein the microporous material comprisesmicropores having a diameter of from about 0.02 microns to about 0.2microns.
 63. The method of claim 51, wherein the microporous materialcomprises aluminum oxide and the pigment comprises an organic dye,wherein the organic dye is covalently attached to the microporousmaterial by a linker comprising an organosilyl group.
 64. The method ofclaim 51, wherein the pigment is deposited on the microporous material.65. The method of claim 64, wherein the pigment comprises an elementalmetal, a metal oxide, a metal alloy, or a combination thereof.
 66. Themethod of claim 65, wherein when the pigment comprises an elementalmetal, the elemental metal comprises a transition metal.
 67. The methodof claim 66, wherein the transition metal comprises palladium, nickel,silver, gold, or a combination thereof.
 68. The method of claim 51,wherein the microporous material comprises aluminum oxide and thepigment comprises one or more elemental transition metals deposited onthe microporous material.
 69. The method of claim 68, wherein theelemental transition metal comprises palladium, nickel, or a combinationthereof.
 70. The method of claim 30, wherein the fluid sample comprisesa biofluid, wherein the biofluid comprises whole blood, serum, plasma,cerebral spinal fluid, urine, saliva, semen, sputum, bronchalveolarlavage fluid, joint aspirate, or wound drainage.
 71. The method of claim30, wherein the fluid sample comprises a bacterium, a virus, a fungus, aspore, a cell culture, a fecal excrement, an animal tissue or cell, avegetable tissue or cell, a lysed ingredient thereof, or a combinationthereof.
 72. The method of claim 30, wherein the fluid sample isprefiltered or treated to remove impurities prior to passing through themicroporous material.
 73. The method of claim 30, wherein the fluidsample further comprises a buffer, a salt, an enzyme, a surfactant, achaotropic agent, or a combination thereof.
 74. The method of claim 30,wherein the fluid sample further comprises NaCl.
 75. The method of claim30, wherein the analyte comprises a virus, a protein, a parasite, afungus, an effector molecule, a ligand, a receptor, a signal-generatingmolecule, a structural molecule, an ion, an antigen, an antibody, atissue, a cell, a component of a cell, a bacterium, a protein, or acombination thereof.
 76. The method of claim 30, wherein the analytecomprises a nucleic acid.
 77. The method of claim 76, wherein thenucleic acid comprises RNA or DNA having at least about 1,500 bases orbase pairs.
 78. The method of claim 76, wherein the nucleic acidcomprises a virion.
 79. The method of claim 78, wherein the virioncomprises an HIV virion.
 80. The method of claim 76, wherein the nucleicacid is labeled with a detectable tracer, wherein the detectable tracercomprises a group comprising a fluorescent microbead, a quantum dot, asurface plasmon resonance particle, a fluorescence generating enzyme, afluorescent dye, or a combination thereof.
 81. The method of claim 76,wherein after step (a), the localized nucleic acid comprises aspecifically localized target nucleic acid.
 82. The method of claim 81,wherein the specifically localized target nucleic acid is labeled with atracer comprising a fluorescent nucleic acid dye.
 83. The method ofclaim 76, wherein after step (a) the localized nucleic acid comprises anon-specifically localized nucleic acid.
 84. The method of claim 83,wherein the non-specifically localized nucleic acid comprises a targetnucleic acid labeled with a specific binding probe comprising afluorescent tracer.
 85. The method of claim 83, wherein thenon-specifically localized nucleic acid comprises a target nucleic acidhybridized with a specific binding probe to form a surface localizedhybrid.
 86. The method of claim 30, wherein at least some of the analyteis labeled either before or after step with a detectable tracer (a) toproduce a labeled analyte.
 87. The method of claim 30, wherein thedetection step (b) is performed by optical detection.
 88. The method ofclaim 86, wherein after step (b), (c) counting the labeled analyte onthe microporous material, and (d) correlating the counted analyte withthe known volume of the fluid sample to determine the analyteconcentration in the fluid sample.
 89. The method of claim 85, whereinthe surface localized hybrid is labeled with fluorescent nucleic aciddye.
 90. The method of claim 76, wherein the nucleic acid is labeledwith a detectable tracer comprising two or more different detectabletracer molecules attached to the nucleic acid.
 91. The method of claim90, wherein the detectable tracer molecules comprise fluorescent tracermolecules.
 92. The method of claim 91, wherein the labeled nucleic acidhas a sufficient fluorescence to achieve a signal-to-noise ratio ofgreater than
 3. 93. The method of claim 91, wherein the labeled nucleicacid has a sufficient fluorescence to achieve a signal-to-noise ratiofrom about 3 to about
 10. 94. The method of claim 86, wherein thelabeled analyte is counted by optical interrogation of a detection areasufficiently small so as to allow none or one labeled analyte to bedetected within the detection area.
 95. The method of claim 86, whereinthe labeled analyte is optically detected with a scanning confocalepifluorometer.
 96. The method of claim 86, wherein the labeled analyteis optically detected with a CCD array based fluorescent device.
 97. Themethod of claim 30, wherein prior to step (a), adding a suspensionmatrix to the fluid sample, wherein the suspension matrix interacts withthe analyte.
 98. The method of claim 97, wherein the suspension matrixcomprises an oligonucleotide, a polysaccharide, a protein, an organic orinorganic polymer or macromolecule, or a combination thereof.
 99. Themethod of claim 97, wherein the suspension matrix comprises calf thymusnucleic acid or salmon sperm nucleic acid.
 100. The method of claim 97,wherein the suspension matrix comprises polyethylene glycol or polyvinylpyrrolidone.
 101. The method of claim 97, wherein the localized analyte,the suspension matrix, or a combination is destabilized.
 102. The methodof claim 30, wherein prior to step (a), localizing a suspension matrixon the microporous material, wherein after step (a), some analyte islocalized on the suspension matrix.
 103. The method of claim 102,wherein the localized analyte, the suspension matrix, or a combinationis destabilized.
 104. A method for amplifying a nucleic acid, comprising(a) passing a fluid sample comprising the nucleic acid through or into amicroporous material, wherein the nucleic acid is localized near thesurface of the microporous material; (b) destabilizing at least some ofthe localized nucleic acid; and (c) amplifying the destabilized nucleicacid.
 105. A method for hybridizing a nucleic acid, comprising (a)passing a fluid sample comprising the nucleic acid through or into amicroporous material, wherein the nucleic acid is localized near thesurface of the microporous material; (b) destabilizing at least some ofthe localized nucleic acid; and (c) hybridizing the destabilized nucleicacid.